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Purification and characterization of Chromobacterium sp. DS-1 cholesterol oxidase with thermal, organic solvent, and detergent tolerance

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Abstract

A new screening method for 6β-hydroperoxycholest-4-en-3-one (HCEO)-forming cholesterol oxidase was devised in this study. As the result of the screening, a novel cholesterol oxidase producer (strain DS-1) was isolated and identified as Chromobacterium sp. Extracellular cholesterol oxidase of strain DS-1 was purified from the culture supernatant. The molecular mass of the purified enzyme was 58 kDa. This enzyme showed a visible adsorption spectrum having peaks at 355 and 450 nm, like a typical flavoprotein. The enzyme oxidized cholesterol to HCEO, with the consumption of 2 mol of O2 and the formation of 1 mol of H2O2 for every 1 mol of cholesterol oxidized. The enzyme oxidized 3β-hydroxysteroids such as cholesterol, β-cholestanol, and pregnenolone at high rates. The K m value for cholesterol was 26 μM. The enzyme was stable at pH 3 to 11 and most active at pH 7.0–7.5, showing optimal activity at pH 7.0 and 65°C. The enzyme retained about 80% of its activity after incubation for 30 min at 85°C. The thermal stability of the enzyme was the highest among the cholesterol oxidases tested. Moreover, the enzyme was more stable in the presence of various organic solvents and detergents than commercially available cholesterol oxidases.

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Acknowledgments

This work was supported in part by the Industrial Technology Research Grant Program in 2005 from New Energy and Industrial Technology Development Organization (NEDO) of Japan, the INOUE ENRYO Memorial Foundation for the Promotion of Sciences, and the Grant for the High Tech Research Center Program organized by Ministry of Education, Culture, Sports, Science and Technology of Japan since 2006. We thank Hiroyuki Ando, Hiromi Kaneko, and Kaori Yoshioka for their technical support.

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Correspondence to Noriyuki Doukyu.

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Doukyu, N., Shibata, K., Ogino, H. et al. Purification and characterization of Chromobacterium sp. DS-1 cholesterol oxidase with thermal, organic solvent, and detergent tolerance. Appl Microbiol Biotechnol 80, 59 (2008). https://doi.org/10.1007/s00253-008-1526-y

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Keywords

  • Cholesterol oxidase
  • Chromobacterium
  • Protein purification
  • Organic solvent
  • Detergent
  • Thermal stability