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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^HNH^OOH + H2CM EC 4.1.99.1) catalyzes a pyridoxal^'-phos- phate-dependent reversible conversion of l- RXH+CH3COCOOH+NH3 (2) tryptophan to indole, pyruvate, and ammonia, RiC^CHNH^OOH + 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 ^^eliminations, 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 in- cubated 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 tryptopha- nase, 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.
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