Symbiobacterium thermophilum is an obligately symbiotic thermophile that can grow only in coculture with a specific Bacillus strain. The amino acid sequences of fragments obtained by cyanogen bromide decomposition of the thermostable beta-tyrosinase (tyrosine phenol-lyase, E.C. 4.1.99.2) from this organism resembled that of the tryptophanase produced by the same organism. DNA-probing with the tryptophanase gene as the hybridization probe led to cloning in Escherichia coli of the beta-tyrosinase (tpl) gene. The nucleotide sequence revealed that the beta-tyrosinase of 458 amino acids (relative molecular mass, 52269) showed significant similarity in amino acid sequence to the tryptophanase over the entire sequence. DNA manipulation of the cloned tpl gene in E. coli led to production of 375 times as much beta-tyrosinase as that produced by the original S. thermophilum strain.

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A thermostable tryptophanase was extracted from a thermophilic bacterium, Symbiobacterium thermophilum strain 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 MonoQ anion-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 enzyme was 4.9. The tryptophanase was stable to heating at 65 degrees C for 20 min and the optimum temperature for the enzyme activity for 20 min reaction was 70 degrees C. The optimum pH was 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 (alpha, beta-elimination) of L-tryptophan into indole, pyruvate, and ammonia in the presence of pyridoxal-5'-phosphate. The Km value for L-tryptophan was 1.47 mM. 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 alpha, beta-elimination of this tryptophanase produced L-tryptophan from indole and pyruvate in the presence of a high concentration of ammonium acetate.