Skip to main content
SpringerLink
Log in

Purification and characterization of extracellular cholesterol oxidase from Enterobacter sp.

  • Original Paper
  • Published:
World Journal of Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

The objective of this work is to obtain an abundant source of cholesterol oxidases for industrial and medicinal needs. Thirteen bacterial strains that express high level of inducible extracellular cholesterol oxidase (COX) were isolated from carnivore feces. One of these strains, named COX8-9, belonging to the genus Enterobacter, was found to produce the highest level of cholesterol oxidase. COX from strain COX8-9 was purified from the culture supernatant by ultrafiltration followed with two consecutive Q-Sepharose chromatographies at different pH values, and then by Superdex-75 gel filtration. The purified enzyme was a monomer with a molecular weight of 58 kDa, and exhibited maximum absorption at 280 nm. The K m value for oxidation of cholesterol by this enzyme was 1.2 × 10−4 M, with optimum activity at pH 7.0. Enzymatic activity of COX was enhanced 3-fold in the presence of metal ion Cu2+, and the enzyme was stable during long-term aqueous storage under various temperatures, indicating its potential as a clinical diagnostic reagent. Preparation and characterization of cholesterol oxidases from the other selected strains are under way.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Aihara H, Watanabe K, Nakamura R (1988) Degradation of cholesterol in egg yolk by Rhodococcus equi no.23. J Food Sci 53:659–660

    Article  CAS  Google Scholar 

  • Allain CC, Poon LS, Chan CSG, Richmond W, Fu PC (1974) Enzymatic determination of total serum cholesterol. Clin Chem 20:470–475

    CAS  Google Scholar 

  • Aono R, Doukyu N, Kobayashi H, Nakajima H, Horikoshi K (1994) Oxidative bioconversion of cholesterol by Pseudomonas sp. strain ST-200 in a water-organic solvent two-phase system. Appl Environ Microbiol 60:2518–2523

    Google Scholar 

  • Buchanan RE, Gibbons NE (1974) Bergey’s manual of determinative bacteriology, Eighth edn. Williams and Wilkins Co., Baltimore, pp 290–340

    Google Scholar 

  • Bru R, Sanchez-Ferrer A, Garcia-Carmona F (1989) Characterization of cholesterol oxidase activity in AOT-isooctane reverse micelles and its dependence on micelle size. Biotechnol Lett 11:237–242

    Article  CAS  Google Scholar 

  • Deeg R, Ziegenhorn J (1983) Kinetic enzymic method for automated determination of total cholesterol in serum. Clin Chem 29:1798–1802

    CAS  Google Scholar 

  • Doukyu N, Aono R (1998) Purification of extracellular cholesterol oxidase with high activity in the presence of organic solvents from Pseudomonas sp. strain ST-200. Appl Environ Microbiol 64:1929–1932

    CAS  Google Scholar 

  • Gadda G, Wels G, Pollegioni L, Zucchelli S, Ambrosius D, Pilone MS, Ghisla S (1997) Characterization of cholesterol oxidase from Streptomyces hygroscopicus and Brevibacterium sterolicum. Eur J Biochem 250:369–376

    Article  CAS  Google Scholar 

  • Isobe K, Mori N, Wakao N (2003) The second cholesterol oxidase produced by gamma-proteobacterium Y-134. J Biosci Bioeng 96:257–261

    CAS  Google Scholar 

  • Kim KP, Rhee CH, Park HD (2002) Degradation of cholesterol by Bacillus subtilis SFF34 isolated from Korean traditional fermented flatfish. Lett Appl Microbiol 35:468–472

    Article  CAS  Google Scholar 

  • Lolekha PH, Jantaveesirirat Y (1992) Streptomyces, a superior source for cholesterol oxidase used in serum cholesterol assay. J Clin Lab Anal 6:405–409

    Article  CAS  Google Scholar 

  • Lolekha PH, Srisawasdi P, Jearanaikoon P, Wetprasit N, Sriwanthana B, Kroll MH (2004) Performance of four sources of cholesterol oxidase for serum cholesterol determination by the enzymatic endpoint method. Clin Chim Acta 339:135–145

    Article  CAS  Google Scholar 

  • MacLachlan J, Wotherspoon AT, Ansell RO, Brooks CJ (2000) Cholesterol oxidase: sources, physical properties and analytical applications. J Steroid. Biochem Mol Biol 72:169–195

    Article  CAS  Google Scholar 

  • Motteran L, Pilone MS, Molla G, Ghisla S, Pollegioni LC (2001) Holesterol oxidase from Brevibacterium sterolicum: the relationship between covalent flavinylation and redox properties. J Biol Chem 276:18024–18030

    Article  CAS  Google Scholar 

  • Richmond W (1973) Preparation and properties of a cholesterol oxidase from Nocardia sp. and its application to the enzymatic assay of total cholesterol in serum. Clin Chem 19:1350–1356

    CAS  Google Scholar 

  • Sampson NS, Vrielink A (2003) Cholesterol oxidases: a study of nature’s approach to protein design. Acc Chem Res 36:713–722

    Article  CAS  Google Scholar 

  • Smith PK, Krohn R, Hermanson GT (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85

    Article  CAS  Google Scholar 

  • Sojo MM, Bru RR, Garcia-Carmona FF (2002) Rhodococcus erythropolis ATCC 25544 as a suitable source of cholesterol oxidase: cell-linked and extracellular enzyme synthesis, purification and concentration. BMC Biotechnol 2:3

    Article  Google Scholar 

  • Srisawasdi P, Jearanaikoon P, Kroll MH, Lolekha PH (2005) Performance characteristics of cholesterol oxidase for kinetic determination of total cholesterol. J Clin Lab Anal 19:247–252

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. College of Life Science, Sichuan Normal University, Chengdu, 610068, P.R. China

    Deping Ye, Jiahong Lei, Wei Li, Fanglan Ge, Ke Wu, Wenliu Xu & Bin Yong

Authors
  1. Deping Ye
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jiahong Lei
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Wei Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Fanglan Ge
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ke Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Wenliu Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Bin Yong
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Wei Li.

Additional information

Deping Ye and Jiahong Lei are contributed equally to this work.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ye, D., Lei, J., Li, W. et al. Purification and characterization of extracellular cholesterol oxidase from Enterobacter sp.. World J Microbiol Biotechnol 24, 2227–2233 (2008). https://doi.org/10.1007/s11274-008-9734-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11274-008-9734-2

Keywords

Search

Navigation