KSBB Journal

The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
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Purification and Characterization of Cholesterol Oxidase Produced by Streptomyces sp. No.4

방선균 Streptomyces sp. No.4가 생산하는 Cholesterol Oxidase의 정제 및 특성

  • 김현수 (계명대학교 자연과학대학 미생물학과) ;
  • 고희선 (계명대학교 자연과학대학 미생물학과)
  • Published : 1999.06.01
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Abstract

The cholesterol oxidase(EC.1.1.3.6) produced from Streptomyces sp. No.4 which isolated from soil was purified and investigated for the enzymatic properties. The enzyme was purified specifically by cholesterol affinity column chromatography with a yield of 28.3%. The purified enzyme showed a single polypeptide on SDS-PAGE and the molecular weight was estimated to be 60,000 daltons. The enzyme activity was strongly inhibited by metal ions such as $HgCl_2$ and $CuSO_4$. Dithiothreitol and mercaptoethanol inhibited the enzyme activity at concentration of 1mM. The Michaelis constant(Km) for cholesterol was found to be 1.38mM by Lineweaver-Burk plot analysis. Amino acid analysis showed that the enzyme protein was composed of 416 amino acid residues including 52moles of glycine and 19moles of tryptophane.

토양에서 분리된 Streptomyces sp으로부터 cholesterol oxidase의 정제 및 효소학적 특성을 조사하였다. 본 효소의 정제는 50~80% 포화의 황산암모늄 침전 및 cholesterol affinity column chromatography를 통하여 28.3%의 수율로 정제 되었다. 정제된 효소는 SDS-PAGE에서 단일한 밴드를 보였으며 분저량은 약 60,000 dalton으로 추정되었으며, HPLC분석 결과 단일의 peak로 검출되었다. 본 효소의 특성을 검토한 결과, 금속 이온으로 Hg와 Cu의 존재시 크게 저해를 받았고, dithiothreitol 과 mercaptoethanol과 같은 저해제에 의해서 상당히 실활되었다. 본 cholesterol oxidase의 Michaelis 상수는 cholesterol을 기질로하여 Lineweaver-Burk plot분석에서 1.38mM로 추산되었다. 정제효소의 아미노산 조정은 약 416개의 잔기로 추정되며 glycine, alanine, threonine의 함량이 높은 반면, methionine, isoleucine의 함량은 극히 낮았다.

Keywords

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