Purification and Properties of Thermostable Tryptophanase from an Obligately Symbiotic Thermophile, Symbiobacterium thermophilum(Microbiology & Fermentation Industry)

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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℃ for 20min and the optimum temperature for the enzyme activity for 20min reaction was 70℃. The optimum pH was 7.0. The NH_2-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 (α,β-elimination) of L-tryptophan into tndole, pyruvate, and ammonia in the presence of pyridoxal-5'-phosphate. The Km value for L-tryptophan was 1.47mM. 5-Hydroxy-L-tryptophan, 5-methyl-DL-tryptophan, L-cystein, S-methyl-L-cystein, and L-serine were also used as substrates and converted to pyruvate. The reverse reaction of α,β-elimination of this tryptophanase produced L-tryptophan from indole and pyruvate in the presence of a high concentration of ammonium acetate.
社団法人日本農芸化学会の論文
1991-12-23

Purification and Properties of Thermostable Tryptophanase from an Obligately Symbiotic Thermophile, Symbiobacterium thermophilum(Microbiology & Fermentation Industry)

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