Purification and Characterization of Cholesterol Oxidase Produced by Streptomyces polychromogenes IFO 13072.

Streptomyces polychromogenes IFO 13072가 생산하는 Cholesterol Oxidase의 정제 및 효소학적 특성

  • 김현수 (계명대학교 자연과학대학 미생물학과) ;
  • 성림식 (계명대학교 자연과학대학 미생물학과) ;
  • 이경화 (계명대학교 자연과학대학 미생물학과) ;
  • 이용직 (계명대학교 자연과학대학 미생물학과) ;
  • 이인선 (계명대학교 자연과학대학 식품가공학과) ;
  • 유대식 (계명대학교 자연과학대학 미생물학과)
  • Published : 2002.06.01

Abstract

Streptomyces polychromogenes IFO 13072 was used as a strain producing cholesterol oxidase(EC 1.1.3.6). The conditions of cholesterol oxidase production were investigated. The optimum composition of medium for production of the enzyme was 1% dextrin, 0.5% casamino acid, 0.1% $KH_2$PO$_4$, 0.5% $NaNO_3$ and 0.05% $MgSO_4$(pH 7.3). The enzyme was purified specifically by cholesterol affinity column chromatography with a yield of 23.2%. The purified enzyme showed a single polypeptide on SDS-PAGE and the molecular weight was estimated about 52,000 daltons. The optimum pH and temperature of the cholesterol oxidase were pH 7.0 and $37^{\circ}C$, respectively. The enzyme was stable in the range of pH 6.0~7.0 and $25^{\circ}C$. The cholesterol oxidase activity was strongly inhibited by metal ions such as $Hg^{2+}$ and $Fe^{2+}$ and inhibitors such as dithiothreitol, mercaptoethanol and isonicotinic acid. The Michaelis constant(Km) for the cholesterol was found to be 25 mM by Lineweaver-Burk plot analysis.

Keywords

Cholesterol oxidase;Streptomyces polychromogenes;producing condition;purification;characterization;cholesterol affinity column chromatography

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