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

Appl Microbiol Biotechnol. 2008 Aug;80(1):59-70. doi: 10.1007/s00253-008-1526-y. Epub 2008 May 30.

Abstract

A new screening method for 6beta-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 3beta-hydroxysteroids such as cholesterol, beta-cholestanol, and pregnenolone at high rates. The Km value for cholesterol was 26 microM. 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 degrees C. The enzyme retained about 80% of its activity after incubation for 30 min at 85 degrees 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / isolation & purification*
  • Bacterial Proteins / metabolism
  • Carbamide Peroxide
  • Cholesterol / metabolism
  • Cholesterol Oxidase / chemistry*
  • Cholesterol Oxidase / genetics
  • Cholesterol Oxidase / isolation & purification*
  • Cholesterol Oxidase / metabolism
  • Chromobacterium / classification
  • Chromobacterium / enzymology*
  • Chromobacterium / genetics
  • Chromobacterium / isolation & purification
  • Drug Combinations
  • Enzyme Stability
  • Hot Temperature
  • Kinetics
  • Molecular Sequence Data
  • Oxidation-Reduction
  • Peroxides / metabolism
  • Phylogeny
  • Solvents / chemistry
  • Substrate Specificity
  • Urea / analogs & derivatives
  • Urea / metabolism

Substances

  • Bacterial Proteins
  • Drug Combinations
  • Peroxides
  • Solvents
  • Carbamide Peroxide
  • Urea
  • Cholesterol
  • Cholesterol Oxidase

Associated data

  • GENBANK/AB426118