PURIFICATION AND PROPERTIES OF THERMOSTABLE BETA-TYROSINASE FROM AN OBLIGATELY SYMBIOTIC THERMOPHILE, SYMBIOBACTERIUM-THERMOPHILUM

Thermostable beta-tyrosinase (tyrosine phenol-lyase, E.C.4.1.99.2) was extracted from an obligately symbiotic and thermophilic bacterium, Symbiobacterium thermophilum, which grows only in co-cultivation with a specific thermophilic Bacillus sp., strain S. The enzyme was purified 300-fold to homogeneity with 4.2% recovery by ammonium sulfate fractionation and several steps of chromatography with anion-exchange, hydroxylapatite, and hydrophobic interaction columns. The enzyme has a molecular weight of approximately 200,000, as estimated by gel filtration column chromatography, and 48,000, as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which indicates that the native enzyme is composed of four homologous subunits. The enzyme was stable up to 80-degrees-C and the optimum temperature for the activity was 80-degrees-C. The enzyme had an optimum pH at 7 and the isoelectric point of pH 4.8. The addition of K+ and NH4+ accelerated the enzyme activity. In contrast, Na+ and Mg2+ showed no effect. The enzyme showed a broad range of substrate specificity, including L-cysteine, D-tyrosine, L-serine, S-methyl-L-cysteine, and beta-chloro-L-alanine. Among them, S-methyl-L-cysteine and beta-chloro-L-alanine were degraded to form pyruvate with higher rates than L-tyrosine. The K(m) values for L-tyrosine, S-methyl-L-Cysteine, and beta-chloro-L-alanine were estimated to be 0.054, 1.67, and 10.2 mM, respectively. Efficient synthesis of 3,4-dihydroxyphenyl-L-alanine (L-DOPA) from pyrocatechol and pyruvate was achieved in the presence of a high concentration of ammonia through the reverse reaction of alpha,beta-elimination by the enzyme.