Summary
A cholesterol-oxidase-producing microorganism, strain COX629, isolated from soil was identified as Pseudomonas sp. The cholesterol oxidase produced by Pseudomonas sp. strain COX629 was purified 2400-fold to homogeneity in an overall yield of 60% from culture broth. The enzyme was a monomer with a molecular weight of 56 000, as estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Sephadex G-150 gel column chromatography. The enzyme showed optimum activity at pH 7.0 and was stable over a rather wide pH range of 4.0 to 11.0. The enzyme showed a high substrate specificity for 3β-hydroxysteroids and the K m value for the oxidation of cholesterol by this enzyme was about 0.2 mM. A characteristic of the enzyme is marked stability at high temperature.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Allain CC, Poon LS, Chan CSG, Richmond W, Fu PC (1974) Enzymatic determination of total serum cholesterol. Clin Chem 20:470–475
Andrews P (1965) The gel-filtration behavior of proteins related to their molecular weights over a wide range. Biochem J 96:595–606
Cowan ST (1974) Characters of Gram-negative bacteria. In: Cowan ST, Steel KJ (eds) Manual for the identification of medical bacteria, 2nd edn. Cambridge University Press, Cambridge, pp 77–122
Fukuyama M, Miyake Y (1979) Purification and some properties of cholesterol oxidase from Schizophyllum commune with covalently bound flavin. J Biochem 85:1183–1193
Inouye Y, Taguchi K, Fujii A, Ishimaru K, Nakamura S, Nomi R (1982) Purification and characterization of extracellular 3β-hydroxysteroid oxidase produced by Streptoverticillium cholesterolicum. Chem Pharm Bull (Tokyo) 30:951–958
Kamei T, Takiguchi Y, Suzuki H, Matsuzaki M, Nakamura S (1978) Purification of 3β-hydroxysteroid oxidase of Streptomyces violascens origin by affinity chromatography on cholesterol. Chem Pharm Bull (Tokyo) 26:2799–2804
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
Palleroni NJ (1984) Genus Pseudomonas. In: Krieg NR, Holt JG (eds), Bergey's manual of systematic bacteriology, vol 1. Williams and Wilkins, Baltimore, pp 141–199
Richmond W (1973) Preparation and properties of bacterial cholesterol oxidase from Nocardia sp. and its application to the enzymatic assay of total cholesterol in serum. Clin Chem 19:1350–1356
Shirokane Y, Nakamura K, Mizusawa K (1977) Purification and some properties of an extracellular 3β-hydroxysteroid oxidase produced by Corynebacterium cholesterolicum. J Ferment Technol 55:337–342
Sih CJ, Wang KC (1965) Mechanism of steroid oxidation by microorganism. J Am Chem Soc 87:1387–1391
Stadtman TC, Cherkes A, Anfinsen CB (1954) Studies on the microbial degradation of cholesterol. J Biol Chem 206:511–523
Starr MP, Stolp H, Truper HG, Balows A, Schlegel HG (1981) The prokaryotes: a handbook on habitats, isolation, and identification of bacteria. Springer, Berlin, Heidelberg, New York
Tomioka H, Kagawa M, Nakamura S (1976) Some enzymatic properties of 3β-hydroxysteroid oxidase produced by Streptomyces violascens. J Biochem 79:903–915
Turfitt GE (1944) The microbiological degradation of steroids. 2. Oxidation of cholesterol by Proactinomyces spp. Biochem J 38:49–62
Uwajima T, Yagi H, Nakamura S, Terada O (1973) Isolation and crystallization of extracellular 3β-hydroxysteroid oxidase of Brevibacterium sterolicum nov. sp. Agric Biol Chem 37:2345–2350
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Lee, Sy., Rhee, Hi., Tae, Wc. et al. Purification and characterization of cholesterol oxidase from Pseudomonas sp. and taxonomic study of the strain. Appl Microbiol Biotechnol 31, 542–546 (1989). https://doi.org/10.1007/BF00270791
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00270791