Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Purification and Characterization of a Cytochrome P-450 from Pravastatin-Producing Streptomyces sp. Y-110.

  • Park, Joo-Woong (Biotechnology Laboratory, Youngjin Research Center, Youngjin Pharmaceutical Co. Ltd.) ;
  • Lee, Joo-Kyung (Biotechnology Laboratory, Youngjin Research Center, Youngjin Pharmaceutical Co. Ltd.) ;
  • Kwon, Tae-Jong (Department of Microbial Engineering, Konkuk University) ;
  • Yi, Dong-Hee (Department of Microbial Engineering, Konkuk University) ;
  • Park, Yong-Il (Environmental Microbiology R.U., Korea Research Institute of Bioscience and Biotechnology) ;
  • Kang, Sang-Mo (Department of Microbial Engineering, Konkuk University)
  • Published : 2001.12.01
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Abstract

Streptomyces sp. Y-110 cytochrome P-450, induced by the addition of compactin -Na into the culture medium, was purified from the cell extract to apparent homogeniety, mainly by DEAE-Sepharose, hydroxyapatite, and Mono Q column chromatyography. The sepcific activity of purified enzyme on its substrate, compactin-Na, was determined to be 15 nmol of pravastatin per mg protein. The molecular mass of this enzyme on SDS-PAGE was $37{\pm}0.5$ kDa, pI was 4.5, and its CO difference spectrum showed maximum absorption peaks at 452 and 550nm, respectively. The N-terminal amino acid sequence was determined to be Met>Thr>Cys>Thr>Pro>Val>Thr>Val>The>Gly>Ala>Ala>Gly>Gln>Ile>Gly>Tyr>Ala>Leu. Its apparent $K_m$ on compactin-Na was $1.294{\mu}M{\cdot}min^-1,\;and\;V_{max}\;was\;1.028{\mu}M{\cdot}min^-1$. The maximum substrate concentration ($K_s$) for reaction was $270 {\mu}M$and thus $1/[K_s]$ was $3.7{\mu}M$. These physicochemical characteristics and kinetic behavior of this enzyme were compared and shown to be different from those of Streptomyces cytochrome P-450 enzymes reported, suggesting that this enzyme may be an additional member of the Streptomyces cytochrome P-450 family.

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