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KMID : 0545120010110061011
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Journal of Microbiology and Biotechnology
2001 Volume.11 No. 6 p.1011 ~ p.1017
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Purification and Characterization of a Cytochrome P-450 from Pravastatin-Producing Streptomyces sp. Y-110
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Park, Joo Woong
Lee, Joo Kyung/Kwon, Tae Jong/Yi, Dong Hee/Park, Yong Il/Kang, Sang Mo
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Abstract
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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 chromatography. The specific activity of purified enzyme on its substrate, compactin-Na, was determined to be 15 n㏖ of pravastatin per ㎎ protein. The molecular mass of this enzyme on SDS-PAGE was 37±0.5 kDa, pI was 4.5, and its CO difference spectrum showed maximum absorption peaks at 452 and 550 ㎚, respectively. The N-terminal amino acid sequence was determined to be Met>Thr>Cys>Thr>Pro>Val>Asn>Val>Thr>Val>The>
Gly>A1a>Ala>Gly>Gln>Ile>Gly>Tyr>Ala>Leu. Its apparent K_m on compactin-Na was 1.294μM·min^-1, and V_max was 1.028μM·min^-1. The maximum substrate concentration (K_s) for reaction was 270μM and thus 1/[K_s] was 3.7μM. 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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