아데노신 탈아미노화 효소 억제제를 생산하는 Streptomyces sp. V-8의 변이종으로부터 페녹사지논 합성효소의 분리 및 특성

Purification and Characterization of Phenoxazinone Synthase from Streptomyces sp. V-8 Mutant Producing Adenosine Deaminase Inhibitor

Phenoxazinone synthase catalyzes the oxidative condensation of two molecules of substituted o-aminophenol to the phenoxazinone chromophore of actinomycin. Mutant strain, Streptomyces sp. V-8-M-1 producing higher phenoxazinone synthase, was obtained from Streptomyces sp. V-8 by treatment of N-methyl-N`-nitro-N-nitrosoguanidine. The phenoxazinone synthase was purified from extract of mutant strain of Streptomyces sp. V-8-M-1 by successive steps of streptomycin sulfate, ammonium sulfate precipitation, DEAE-cellulose and Sephadex G-200 column chromatography. Molecular weight of the enzyme was 360,000 daltons. The enzyme was composed of octamer of a single subunit of 45,000daltons. The Km value and Vmax value for 3-HAA were 14.9mcM and 9.5mg/U, respectively. The optimal pH and temperature for the enzyme activity were 9.0 and 25-30oC, respectively. Treatment of the enzyme with group specific reagents, phenylglyoxal, p-hydroxymercury-benzoate, N-bromosuccinimide, 5,5`-dithiobis-nitrobenzoic acid and ethylmaleimide resulted in loss of enzyme activity, which shows arginine and cysteine residues are at or near the active site.