Purification and Properties of Thermostable

Agric. Biol. Chem., 55 (12), 3059-3066, 1991 3059

Purification and Properties of Thermostable Tryptophanase from an Obligately Symbiotic Thermophile, Symhiobacterium thevmophilum Seibun Suzuki, Toshikatsu Hirahara, Sueharu Horinouchi and Teruhiko Beppu* Department of Agricultural Chemistry, Faculty of Agriculture, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113, Japan Received June 28, 1991

A thermostable tryptophanase was extracted from a thermophilic bacterium, Symbiobacterium thevmophilumstrain T, which is obligately symbiotic with the thermophilic Bacillus strain S. The enzyme was purified 21-fold to homogeneity with 19%recovery by a series of chromatographies using anion-exchange, hydroxylapatite, hydrophobic interaction, and MonoQanion-exchange columns. The molecular weight of the purified enzyme was estimated to be approximately 210,000 by gel filtration, while the molecular weight of its subunit was 46,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which indicates that the native enzyme is composed of four homologous subunits. The isoelectric point of the enzymewas 4.9. The tryptophanase was stable to heating at 65°C for 20min and the optimumtemperature for the enzyme activity for 20min reaction was 70°C. The optimum pHwas 7.0. The NH2-terminal amino acid sequence of this tryptophanase shows similarity to that of Escherichia coli K-12, despite a great difference in the thermostability of these two enzymes. The purified enzyme catalyzed the degradation (a,/J-elimination) of L-tryptophan into indole, pyruvate, and ammoniain the presence of pyridoxal-5'-phosphate. The Kmvalue for L-tryptophan was 1.47mM. 5-Hydroxy-L-tryptophan, 5-methyl-DL-tryptophan, L-cysteine, S-methyl-L-cysteine, and L-serine were also used as substrates and converted to pyruvate. The reverse reaction of a,/J-elimination of this tryptophanase produced L-tryptophan from indole and pyruvate in the presence of a high concentration of ammoniumacetate.

Tryptophanase (L-tryptophan indole-lyase, RiCH^HNHÔOH + H2CM EC 4.1.99.1) catalyzes a pyridoxal^'-phosphate-dependent reversible conversion of l- RXH+CH3COCOOH+NH3 (2) tryptophan to indole, pyruvate, and ammonia, RiC^CHNHÔOH + R2H^> which is called an a,/?-elimination reaction R2CH2CHNH2COOH + RXH (3) (Reaction I).1-2* The catalytic mechanism of tryptophanase as L-Tryptophan + H2O+± a typical memberof the pyridoxal phosphate- Pyruvate + Indole + NH3 (1) dependent enzymes has been extensively At high substrate concentrations, tryptopha- studied.5'6) Since the a,/?-elimination reaction nase catalyzes many other ^êliminations, itself is reversible under appropriate conditions, as well as numerous ^-replacement reactions tryptophanase can be used as a catalyst to (Reactions 2 and 3), in which Rx represents synthesize L-tryptophan from indole, pyruvate, -OH, -OCH3, -SH, -SCH3, or indolyl radicals and ammonia.7) For example, Nakazawa et al.8'9) suggested that L-tryptophan and 5- and R2 represents indolyl radicals.3'4) hydroxy-L-tryptophan might be economically

Corresponding author. 3060 S. Suzuki et al.

produced from synthetic starting materials culture were harvested using a continuous-flow centrifuge such as sodium pyruvate, indole, and 5- at 27,000xg, washed twice with 100ml of 0.1m sodium phosphate buffer, pH 7.0, containing 3% sodium citrate, hydroxy indole by the tryptophanase-contain- and then suspended in 50ml of the same buffer. To ing cells of Proteus rettgeri. obtain cells of S. thermophilum almost free from the Tryptophanases have been found mainly in Bacillus cells, lysozyme and EDTAwere added to the the Gram-negative bacterial species indigenous suspension to give final concentrations of 300/ig/ml and to the intestinal tracts of animals, but very few 200/ig/ml, respectively, and the mixture was incubated at species of other categories.10" 12) In the course 35°C for about 15min. By this procedure the cells of Bacillus strain S were almost completely lysed, while the of a screening program for thermostable cells of S. thermophilum remained almost intact. The tryptophanases as a potential industrial cata- remaining cells were centrifuged at 13,000 x g for 20min lyst for L-tryptophan synthesis, we had found and washed three times with 100ml of 50mMpotassium that a mixed culture of thermophiles estab- phosphate buffer, pH 6.8, containing 10/im pyridoxal-5'- lished from a compost heap produced both phosphate, 1 mM2-mercaptoethanol, and 0.25 mMphenyl- heat-stable tryptophanase and /?-tyrosinase. methylsulfonyl fluoride (PMSF). The same buffer was used in the following purification procedure as the standard Interestingly these two thermostable enzyme buffer. Pellets were stored at -20°C until use. Approx- activities were expressed by a hitherto un- imately 1.75g of cells (wet weight) was obtained from knownbacterium, Symbiobacterium thermo- 1 liter of the culture. philum strain T, which absolutely requires co-culture with a specific thermophilic Bacillus Assay of tryptophanase and fl-tyrosinase. Tryptophanase strain S.13) In this paper, we describe the activity was assayed by measuring the amountof indole or pyruvate formed from L-tryptophan. The reaction purification of the thermostable tryptophanase mixture contained in a total volume of 4ml: 2.5mM from S. thermophilum strain T and some L-tryptophan; 50mM potassium phosphate buffer, pH properties of the tryptophanase. This trypto- 8.0; 0.1 mMpyridoxal-5'-phosphate, and an appropriate phanase has also been found to be useful for amount of enzyme preparations. The mixture was incubated at 65°C for 20min, and the reaction was stopped enzymatic production of L-tryptophan. by the addition of 1 ml of 30% trichloroacetic acid. The indole formed was measured by the method of McEvoy.14) Materials and Methods For examination of the substrate specificity of tryptophanase, the pyruvate formed from various substrates was Microorganisms. A lyophilized mixed culture of S. measured by the method of Friedemann and Haugen.15) thermophilum strain T and Bacillus strain S was used as One unit of the enzymeactivity was defined as the amount the seed culture. A basal mediumnamed PEP medium of enzyme catalyzing the formation of 1 fimol of product consisted of 0.2% each of L-tryptophan and L-tyrosine, per min under the assay conditions described above. The 0.5% Polypepton (Daigo Chemicals), 0.1% Bacto yeast specific activity was expressed as the units of enzyme extract (Difco), 0.3 % K2HPO4, 0.1% KH2PO4, 0.05% activity per mgof protein. Protein concentrations were MgSO4à"7H2O, and 0.05% pyridoxal-5'-phosphate. l- measured with a Bio-Rad protein assay kit using bovine Tryptophan and L-tyrosine, which are essentially required serum albumin as the standard.16>17) for the induction of the tryptophanase and /?-tyrosinase, /?-Tyrosinase activity was measured by the amount of respectively, were separately autoclaved at 115°C for pyruvate formed from L-tyrosine. The reaction mixture 15min and added to the medium. contained in a total volume of 4ml: 50mM potassium phosphate buffer (pH 8.0), 2.5mM L-tyrosine, 0.1mM Culture. Lyophilized cells of S. thermophilum strain T pyridoxal-5'-phosphate, and an appropriate amount of and Bacillus strain S were inoculated into 200ml of PEP medium in a 500-ml Erlenmeyer flask and cultured at enzymepreparations. 60°C for 24^30hr without shaking. Five ml of the seed Sodium dodecyl sulfate-polyacrylamide gel electropho- culture was inoculated into 1 liter of PEP medium and resis (SDS-PAGE). Slab gel electrophoresis under denatur- cultured at 60°C in a 5-liter Erlenmeyer flask without ing conditions was done as described by Laemmli18'19) shaking. Bacillus strain S started to grow at first. S. with 12.5% polyacrylamide gels with a 1-mm thickness. thermophilum then started to grow when Bacillus strain S Protein bands were fixed and stained with Coomassie began to lyse at its early stationary phase. After about brilliant blue R-250 in 25% isopropanol-10% acetic acid. 30hr, the cell concentration of S. thermophilum reached approximately 2 x 1O10cells/ml, while that of strain S was Molecular mass measurement.The relative molecular about 1 x 107cells/ml. The cells from 12liters of such a mass of the enzyme was measured by gel filtration on a Thermostable Tryptophanase from S. thermophilum 3061

Sephacryl S-300 column. After the purified tryptophanase oligomeric structure of the enzyme at a low had been put on the column (2.6x58cm) with a jacket to keep the temperature around 35°C, the enzyme was temperature. 1) Preparation of cell extracts. Frozen cells eluted with the standard buffer containing 200mMKC1 of S. thermophilum (about 21 g wet weight) at a flow rate of 17ml/hr. The standard proteins used for calibration were bovine serum albumin (67 kilodaltons, were suspended in 60ml of the standard buffer kDa), aldolase (158 kDa), and catalase (232kDa). described above and disrupted by sonication SDS-PAGEwas also used for the measurement of the with a Branson Sonifler. Cell debris was relative molecular mass of a subunit. The reference proteins used were lysozyme (14.4kDa), soybean trypsin removed by centrifugation at 25,000 xg for inhibitor (20.1 kDa), carbonic anhydrase (30kDa), oval- 30min and streptomycin sulfate (Wako Pure bumin (43kDa), bovine serum albumin (67kDa), and Chemicals, Osaka) was added at a final phosphorylase b (94 kDa). concentration of 1 mg/ml to remove nucleic acids as precipitates by centrifugation at Isoelectric point measurement. The isoelectric point (pi) 25,000 xg for 30min. was measured with polyacrylamide gels using a Pharmacia FBE 3000 electrophoresis kit. Ampholine was used to 2) DEAE-TOYOPEARLcolumn chromatog- generate a pH gradient from pH 4 to 6.5. After raphy. The supernatant was put directly on a electrophoresis at 4°C overnight at a constant power of 6 DEAE-Toyopearl 650M column (4.1 x 9.8 cm) watts, the gels were fixed with 5% sulfosalicylic acid and previously equilibrated with the standard 10% trichloroacetic acid, and stained with Coomassie brilliant blue R-250. The reference proteins used were buffer described above. After the column had egg albumin (pi 4.15), soy bean trypsin inhibitor (pi been washed with 150ml of the standard 4.55), /Mactoglobulin A (pi 5.20), and bovine carbonic buffer, proteins were eluted with a linear anhydrase B (pi 5.85). gradient ofKC1 from 0 to 0.4m in the standard buffer at a flow rate of 30ml/hr with a fraction NH2-terminal amino acid sequencing. For identification size of 5ml. The tryptophanase activity was of the NH2-terminal amino acid sequence, the purified eluted at approximately 190mM KC1. The tryptophanase was put through automatic Edman degradation by an automated protein sequencer (Applied active fractions were combined and con- Biosystems, 470 A) or a spinning-cup sequenator centrated by ultra filtration through a PM10 (Beckmann, 890 C). The amount of samples used were filter (Amicon Corp.). The concentrated pro- 100/zg and 500fig, respectively. Phenylthiohydantoin tein solution was dialyzed against the standard derivatives of amino acids were identified and measured buffer. by gas-liquid chromatography or high-performance liquid chromatography. 3) Hydroxylapatite column chromatography. The above preparation was put on a hydroxyl- Analytical method. L-Tryptophan was analyzed by apatite column (3.65 x ll.1 cm; Bio-Gel HT) HPLC equipped with a column of /^Bondasphere 5/iC18- previously equilibrated with the standard 100A (Waters Limit.; 3.9mmx 150mm). Elution was buffer. Proteins were eluted with a linear done with 50%methanol in 20mMsodium acetate buffer (pH 4.0). L-Tryptophan was detected by fluorometry gradient of 50-300 mMpotassium phosphate by measuring the emission fluorescence at 355nm with in the standard buffer in a total volume of excitation at 275nm. 400ml. The tryptophanase activity was eluted from the column at HOmMpotassium phosphate, as shown in Fig. 1. The peak fractions Results and Discussion were combined, concentrated with a PM10 Purification of tryptophanase filter, and dialyzed against the standard buffer. The thermostable tryptophanase was puri- 4) Hydrophobic interaction column chroma- fied from the cell extract of S. thermophilum tography. Ammoniumsulfate was added at a by a series of chromatographies as described final concentration of 0.8m to the enzyme below. This procedure was established to preparation from step 3. After the mixture purify tryptophanase and jS-tyrosinase simul- was left overnight at 4°C, the supernatant taneously. All operations were done at room was obtained by centrifugation at 13,000xg temperature to avoid dissociation of the for 20min. The supernatant was clarified 3062 S. Suzuki et al.

Fig. 1. Separation of the Tryptophanase by Bio-Gel HTChromatography. Conditions are described in the text. The tryptophanase activity (#) was eluted at HOniM potassium phosphate. by passage through a 0.22-/im nylon filter cartridge and then chromatographed on a Phenyl-Superose HR5/5 column (5 x 50mm; Pharmacia LKB Biotechnology) previously equilibrated with the standard buffer containing 0.8 m ammoniumsulfate. The column was equipped on a Pharmacia fast protein liquid chromatography (FPLC) system, and proteins were eluted with simultaneous linear gradients of 0.8-0m ammoniumsulfate and 0-40% (vol/vol) ethyleneglycol at a flow rate of 0.5ml/min in the standard buffer in a total volume of 30ml. The active fractions were combined, concentrated, and dialyzed against the standard buffer. 5) MonoQanion-exchange chromatography. The enzymepreparation was finally chromatographed over a MonoQ HR 5/5 anion- Fig. 2. Separation of the Tryptophanase by MonoQ exchange column (0.5 x 5 cm; Pharmacia LKB) Anion-exchange Chromatography. using the FPLC system. The tryptophanase Conditions are described in the text. The elution profile is was eluted with a linear gradient of 0-0.4m shown in the upper half. The fractions containing the tryptophanase (TPase) and the /?-tyrosinase (TYase) are KC1 in the standard buffer of a total 60ml at indicated. The SDS-PAGEprofile of the combined active a flow rate of l.Oml/min. The elution profile fractions is shown in the bottom half. Lane 1 contains the is shown in Fig. 2A. The tryptophanase was relative molecular mass standards and lane 2 contains the separated from the /?-tyrosinase at this step purified tryptophanase. Thermostable Tryptophanase from S. thermophilum 3063

Table I. Summary of Purification of Tryptophanase from S. thermophilum

P urifi tion ste Totalprotein Totalunit Specific activity Purification Yield (mg) (U) (U/mg) (fold) (%)

Crude extract 1,185 498 0.364 1 100 DEAE-Toyopearl 255.3 249.2 0.976 2.7 50 Hydroxylapatite 51.5 170.5 3.308 9. 1 34 Phenyl-Superose (FPLC) 21.1 126.3 5.98 16.4 25 MonoQ (FPLC) 12.6 96.1 7.60 20.9 19

Fig. 3. Comparison of the NH2-Terminal Amino Acid Sequences of the Tryptophanases from S. thermophilum and E. coli K-12. Identical amino acids (*) and similar amino acids (+) are indicated. X represents an ambiguous residue. and the enzymatic properties of the /?- Gly-Glu-Pro-Phe-Lys-Ile-Lys-Met-Val-Glu- tyrosinase will be reported elsewhere. The Pro-Ile-Arg-Leu-Ile-Pro-Arg-Glu-Asp-Arg- fraction containing tryptophanase activity was Glu-Ala-Ala-Ile-Lys-Ala-Ala-Y-Tyr-Asn-Pro- then dialyzed against the standard buffer and Phe-Leu- (where X represents Pro or Ser, and stored at 4°C until use. The final preparation Y represents an ambiguous residue). This showed a single protein band on SDS-PAGE sequence shows a similarity to that of the as shown in Fig. 2B. The results of the whole tryptophanase from E. coli K-12 (Fig. 3),25'26) purification process are shown in Table I. The despite a big difference in thermal stability. overall procedure gave 21-fold purification with 19%recovery. Isoelectric point The isoelectric point (pi) of the trypto- Molecularmassmeasurementoftryptophanase phanase was 4.9, when the marker proteins The relative molecular weight of the trypto- with p/s of 4.15, 4.55, 5.20, and 5.85 were phanase subunit was estimated to be 46,000 Da used as references. by SDS-PAGE (data not shown). The molecular massof the native enzymewas estimated Optimum temperature and heat stability of to be 210,000 Da by gel filtration. We therefore tryp tophanase concluded that this tryptophanase was com- The enzymeactivity was measured at various posed of four identical subunits, which is temperatures from 30 to 85°C. The optimum consistent with the observations that all the temperature of the S. thermophilum tryptopha- tryptophanases reported so far are composed nase activity was 70°C, when the standard of four identical subunits. The molecular mass reaction mixtures containing L-tryptophan as of the subunit was 46,000Da which was the substrate were incubated for 20min (Fig. slightly smaller than those from E. coli (Mr 4A). This is distinctly higher than that of the 55,000 Da),4) Bacillus alvei (Mr 52,000 Da),17) E. coli tryptophanase (55°C; obtained from and P. rettgeri (Mr 55,000Da).21~24) Sigma Chem. Co.). The thermostability of the tryptophanase was examined by incubating the NH2-Terminal amino acid sequence enzymefor 20min at various temperatures Automated Edman degradation established (Fig. 4B). The S. thermophilum tryptophanase the NH2-terminal amino acid sequence of the was found to be stable up to 65°C in contrast S. thermophilum tryptophanase to be X-Lys- to the E. coli tryptophanase which was 3064 S. Suzuki et al.

Fig. 4. Effects of Temperature on the Activity (A) and Thermostability (B) of the S. thermophilum Tryptophanase Fig. 5. Effects of pH on the Activity (A) and Stability (#) and the E. coli Tryptophanase (A)- (B) of the S. thermophilum Tryptophanase. (A): The standard reaction mixture containing 0.01 units (A): The standard reaction mixture containing 0.01 units of the enzyme was incubated at various temperatures for of the enzyme was incubated at 65°C at various pHs. 20min and the amounts of indole formed were measured The reaction mixture contained: 2.5 mMof L-tryptophan, by the method of McEvoy-Bowe.14) 0.1mM of pyridoxal phosphate and 0.01units of the (B): After heat-treatment of the enzymes at various enzyme, in a total volume of 4ml of the following buffer temperatures for 20min, the remaining activity was (0.05 m each); potassium phosphate buffer (O), glycine- measured by reaction at 65°C for 20min and expressed as NaOHbuffer (A), glycine-NaOH buffer containing 5 mM a percentage of the activity without the heat-treatment. ammoniumsulfate (æf), glycine-NaOH buffer containing 10mMKC1(A), citrate sodium phosphate buffer (#), Tris-HCl buffer (å¡), and NaHCO3-NaOHbuffer (å ). completely inactivated at the same tempera- (B): The enzyme was incubated for 48hr at 25°C at ture. The specific activity of the purified S. various pHs, and the remaining activity was measured thermophilum tryptophanase was 7.6 at 65°C, by reaction at 65°C for 20min in 0.05m potassium and below 0.5 at 30°C, while those of P. phosphate buffer (pH 8.0) and expressed as a percentage rettgeri and E. coli tryptophanases were of the activity without any pHtreatment. reported to be 9.0 at 30°C,21-23) and 26.0 at 37°C.6) These properties of the S. thermophilum OptimumpH and pH stability enzymeare expected to be useful for industrial The effects of pH on the activity of uses. tryptophanase were examined. The tryptophanase had its optimum pH for activity at 7.0 (Fig. 5A) and it was quite stable between pH Thermostable Tryptophanase from S. thermophilum 3065

6.0 and 10.0 (Fig. 5B). The tryptophanase Table II. Substrate Specificity of Tryptophanase. All assays were run in 50mMpotassium phosphate activity was very low when the glycine-NaOH buffer (pH 8.0) containing 0.068 units of the enzyme, buffer was used, probably because the trypto- 0.1 mMof pyridoxal-5'-phosphate, and 25mMof each phanase was activated by monocations such substrate. The amount ofpyruvate formed was measured asNH4+ and K+. by the method of Friedemann and Haugen15) with 2,4-dinitro-phenylhydrazine. Substrate specificity Substrates Relative rate (%) The relative rates of pyruvate formation L-Tryptophan 1 00 from various amino acid derivatives by the S-Methyl-L-cysteine 63.8 action of this tryptophanase are presented in L-Cysteine 18.9 Table II. In comparison with L-tryptophan as 5-Methyl-DL-tryptophan 1 5.2 the substrate, S-methyl-L-cysteine showed a 5-Hydroxy-L-tryptophan 6.7 L-Serine 5.3 relatively high reactivity, which was approx- D-Tryptophan 0 imately 63.8% of that with L-tryptophan. L-Tyrosine 0 Pyruvate formation was also observed with l- D-Tyrosine 0 cysteine, 5-methyl-DL-tryptophan, 5-hydroxy- D-Serine 0 L-Homoserine 0 L-tryptophan, and L-serine but at lower rates. L-Threonine 0 D-Tryptophan and other D-amino acids were L-Methionine 0 not substrates of this tryptophanase. These L-Phenylalanine 0 results indicated that the S. thermophilum tryptophanase also catalyzed a,/?-elimination of various L-amino acids in addition to L-tryptophan. The substrate specificity of the S. thermophilum tryptophanase is similar to that of the P. rettgeri tryptophanase.

Calculation of the Michaelis constant The Km value of the tryptophanase for L-tryptophan was calculated to be 1.47mM based on a Lineweaver-Burk reciprocal plot (Fig. 6). This value is distinctly higher than those of the E. coli tryptophanases (0.33 mM)4) and the P. rettgeri enzyme (0.26mM).24) The Fig. 6. Lineweaver-Burk Plot of the Tryptophanase Reaction with L-Tryptophan as the Substrate. Fmaxand kcat were calculated to be 13.7 The reaction mixture contained: 50mM of potassium /miol/min/mg enzyme and 2520 min phosphate buffer (pH 8.0), 0. 1 him ofpyridoxal phosphate, -l 0.027 units of the enzyme, and variable amounts of Synthesis of L-tryptophan by the reversal of L-tryptophan in a total volume of 4ml. The enzyme ^-elimination of the tryptophanase activity was measured by the amount of indole formed Weexamined the possibility of synthesizing from L-tryptophan. L-tryptophan from indole, ammonia, and pyruvate by the reverse reaction of this decomposition of pyruvate during prolonged tryptophanase. The reaction conditions were incubation. When indole and pyruvate were essentially based on the data of Yoshida et incubated with the tryptophanase in the ^ 23,24) Although the optimum temperature presence ofa high concentration ofammonium of the enzyme was 70°C in the former acetate, L-tryptophan was synthesized as a experiment, a lower temperature of 30°C was function of the incubation period, as shown in used for the synthesis to avoid gradual Fig. 7. After 50 hr of incubation, L-tryptophan inactivation of the enzyme and thermal was synthesized with a 75% yield from indole. 3066 S. Suzuki et al.

thermostable tryptophanase gene in some appropriate host are required.

References 1) W. A. Wood, I. C. GunsalusandW. W. Umbreit, /. Biol. Chem., 170, 313 (1947). 2) H. Kumagai, H. Yamada, H. Matsui, H. Ohgishi and K. Ogata, /. Biol. Chem., 245, 1767 (1970).. 3) W. A. Newton, Y. Morino and E. E. Snell, /. Biol. Chem., 240, 1211 (1965). 4) W. A. NewtonandE. E. Snell, Proc. Natl. Acad. Sci. U.S.A., 51, 382 (1964). Fig. 7. Synthesis of L-Tryptophan from Pyruvate, 5) Y. Morino and E. E. Snell, /. Biol. Chem., 242, Ammonia, and Indole by the Reverse Reaction of 2793, 2800, 5591, 5602 (1967). a,/?-Elimination of the S. thermophilum Tryptophanase. 6) E. E. Snell, Adv. Enzymol., 42, 287 (1975). 7) T. Watanabe and E. E. Snell, Proc. Natl. Acad. Sci. The initial reaction mixture in a total volume of 50ml U.S.A., 69, 1086 (1972). contained 50mM potassium phosphate buffer (pH 8.1), 8) H. Nakazawa, H. Enei, S. Okumura, H. Yoshida 0.4mM pyridoxal-5'-phosphate, 25mM pyruvate, 25mM indole, 650mM ammonium acetate, 8mM EDTA, 8mM and H. Yamada, FEBS Lett., 25, 43 (1972). sodium sulfite, 1% Triton X-100, and 5.07 units of the S. 9) H. Nakazawa, H. Enei, S. OkumuraandH. Yamada, thermophilum tryptophanase. The reaction was done at Agric. Biol. Chem., 36, 2523 (1972). 30°C. The amounts of L-tryptophan (0) synthesized from 10) A. B. Pardee and L. S. Prestidge, Biochim. Biophys. pyruvate (A) and indole (O) are indicated. Ada, 49, 71 (1961). ll) J. A. Hoch and R. D. DeMoss, /. Bacteriol., 90, 604 (1965). Agreater decrease in the amountof pyruvate 12) R. D. DeMoss and K. Moser, /. Bacteriol, 98, 167 was probably due to thermal decomposition. (1969). Because of the high thermostability of the 13) S. Suzuki, S. Horinouchi and T. Beppu, J. Gen. Microbiol, 143, 2353 (1988). tryptophanase, no significant decrease in the 14) B. E. McEvoy, Analyst., 88, 893 (1963). enzyme activity was observed even after 50 hr 15) T. E. Friedemann and G. E. Haugen, /. Biol. Chem., of incubation (data not shown). 147, 415 (1943). In the case of the P. rettgeri tryptophanase, 16) M. M. Bradford, Anal. Biochem., 72, 248 (1976). the equilibrium between degradation and 17) J. J. Sedmak and S. E. Grossberg, Anal. Biochem., synthetic reactions inclined toward the syn- 79, 544 (1977). 18) P. H. O'Farrell, /. Biol. Chem., 250, 4007 (1975). thetic state at a level of approximately 80 19) U. K. Laemmli, Nature, 227, 680 (1970). molar%.9) Wetherefore suppose that the rate 20) J. A. Hochand R. D. DeMoss, /. Biol. Chem., 247, of 75molar% conversion of indole into l- 1750 (1972). tryptophan obtained by the above synthetic 21) H. Yoshida, T. Utagawa, H. Kumagai and H. reaction is satisfactory, although we have not Yamada, Agric. Biol. Chem., 38, 2065 (1974). measured the equilibrium of a,/?-elimination 22) H. Yoshida, H. Kumagai and H. Yamada, Agric. Biol. Chem., 38, 2073 (1974). reaction by the S. thermophilum tryptophanase. 23) H. Yoshida, H. Kumagai, H. Yamada and H. These results indicate that the tryptophanase Matsubara, Biochim. Biophys. Ada, 391, 494 (1975). from an obligately symbiotic thermophile, 24) H. Yoshida, H. Kumagai and H. Yamada, Agric. S. thermophilum, is a potential catalyst for Biol. Chem., 38, 463 (1974). enzymatic production of L-tryptophan. To 25) H. Kagamiyama, H. Matsubara and E. E. Snell, /. overcome the difficulty of preparing the en- Biol. Chem., 247, 1576 (1972). 26) M. C. Deeley and C. Yanofsky, J. Bacteriol., 141, zyme from the symbiotic mixed cultivation of 787 (1981). this organism, cloning and expression of the