Skip to main content
SpringerLink
Log in

Coenzyme precursor-assisted expression of a cholesterol oxidase from Brevibacterium sp. in Escherichia coli

  • Original Research Paper
  • Published:
Biotechnology Letters Aims and scope Submit manuscript

Abstract

The gene (choB b ), encoding cholesterol oxidase from Brevibacterium sp. CCTCC M201008, was cloned and sequenced by PCR (GenBank accession number: DQ345780). The gene consists of 1653 base pairs and encodes a protein of 551 amino acids. ChoB b exhibited a homology of 98% with cholesterol oxidase gene from Brevibacterium sterolicum ATCC 21387. The cholesterol oxidase gene, cloned in the vector pET-28a, was over-expressed in Escherichia coli BL21–CodonPlus (DE3)-RP grown at 23°C in Luria-Bertani medium containing 50 μM riboflavin, the precursor of the FAD coenzyme of the enzyme. A maximum activity of 3.7 U/mg was obtained from cell free extract of E. coli BL21-CodonPlus (DE3)-RP harboring the pET-28a-choBb.

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

Similar content being viewed by others

Reference

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

    PubMed  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

    PubMed  Google Scholar 

  • Cecchini G, Perl M, Lipsick J, Singer TP, Kearney EB (1979) Transport and binding of riboflavin by Bacillus subtilis. J Biol Chem 254:7295–7301

    PubMed  CAS  Google Scholar 

  • Cho HJ, Choi KP, Yamashita M (1995) Introduction and expression of the Streptomyces cholesterol oxidase gene (choA), a potent insecticidal protein active against Boll weevil larvae, into tobacco cells. Appl Microbiol 44:133–138

    CAS  Google Scholar 

  • Ishizaki T, Hirayama N, Shinkawa H, Nimi O, Murooka Y (1989) Nucleotide sequence of the gene for cholesterol oxidase from a Streptomyces sp. J Bacterol 171:596–601

    CAS  Google Scholar 

  • Kreit J, Lefebvre G, Germain P (1994) Membrane-bound cholesterol oxidase from Rhodococcus sp. cells; production and extraction. J Biotechnol 33:271–282

    Article  CAS  Google Scholar 

  • Liu W, Hsu J, Wang W (1983) Production of cholesterol oxidase by antibiotic resistant mutant and a constitutive mutant Arthrobacter simplex B-7. Proc Natl Sci Counc ROC 7:255–260

    CAS  Google Scholar 

  • Lv CF, Wang W, Tang YX, Wang LG (2002) Effect of cholesterol bioavailability- improving factors on cholesterol oxidase production by a mutant Brevibacterium sp. DGCDC-82. Process Biochem 37:901–907

    Article  Google Scholar 

  • Murooka Y, Ishizaki T, Nimi O, Maekawa N (1986) Cloning and expression of a Streptomyces cholesterol oxidase gene in Streptomyces lividans with plasmid pIJ702. Appl Environ Microbiol 52:1382–1385

    PubMed  CAS  Google Scholar 

  • Ohta T, Fujishiro K, Yamaguchi K, Tamura Y, Aisaka K, Uwajima T et al. (1991) Sequence of gene choB encoding cholesterol oxidase of Brevibacterium sterolicum: comparison with choA of Streptomyces sp. Gene 103:93–96

    Article  PubMed  CAS  Google Scholar 

  • Purcell JP, Greenplate JT, Jennings MG (1993) Cholesterol oxidase: a potent insecticidal protein active against Boll weevil larvae. Biochem Biophys Res Commun 196:1406–1413

    Article  PubMed  CAS  Google Scholar 

  • Richmond W (1992) Analytical reviews in clinical biochemistry: the quantitative analysis of cholesterol. Ann Clin Biochem 29:577–597

    PubMed  CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, NY

    Google Scholar 

  • Sampson NS, Chen X (1998) Increased expression of Brevibacterium sterolicum cholesterol oxidase in Escherichia coli by genetic modification. Protein Expr Purif 12:347–352

    Article  PubMed  CAS  Google Scholar 

  • Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467

    Article  PubMed  CAS  Google Scholar 

  • Shirokano Y, Nakamura K, Mizusawa K (1977) Purification and some properties of an extracellular 3b-hydroxy steroid oxidase produced by Corynebacterium cholesterolicum. J Ferment Technol 55:337–346

    Google Scholar 

  • Sojo M, Bru R, Lopez-Molina D, Garcia-Carmona F, Argulles JC (1997) Cell-linked and extracellular cholesterol oxidase activities from Rhodococcus erythropolis, isolation and physiological characterization. Appl Microbiol Biotechnol 47:583–589

    Article  PubMed  CAS  Google Scholar 

  • Tomioka H, Kagawa M, Nakamura S (1976) Some enzymatic properties of 3b-hydroxy steroid oxidase produced by Streptomyces violascens. J Biochem 79:903–915

    PubMed  CAS  Google Scholar 

  • Uwajima T, Yagi H, Terada O (1974) Properties of crystalline 3β-hydroxysteroid oxidase of Brevibacterium sterolicum. Agric Biol Chem 38:1149–1156

    CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Key Laboratory of Industrial Biotechnology of Ministry of Education and School of Biotechnology, Southern Yangtze University, Wuxi, 214036, P.R. China

    Longgang Wang & Wu Wang

Authors
  1. Longgang Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Wu Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Longgang Wang.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wang, L., Wang, W. Coenzyme precursor-assisted expression of a cholesterol oxidase from Brevibacterium sp. in Escherichia coli . Biotechnol Lett 29, 761–766 (2007). https://doi.org/10.1007/s10529-006-9295-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10529-006-9295-0

Keywords

Search

Navigation