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

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

  • 김경자 (순천향대학교 자연과학대학 생명과학부) ;
  • 조성진 (순천향대학교 자연과학대학 생명과학부)
  • Published : 1999.02.01

Abstract

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-l 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 45,000 daltons. The Km value and Vmax value for 3-HAA were $14.9{\;}{\mu}M$ and 9.5 mg/U, respectively. The optimal pH and temperature for the enzyme activity were 9.0 and $25~30^{\circ}C$, respectively. Treatment of the enzyme with group specific reagents, phenylglyoxal, p-hydroxymercury-benzoate, Nbromosuccinimide, 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.

Keywords

References

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