Skip to main content
SpringerLink
Log in

Expression, purification and functional characterization of a recombinant 2,3-dihydroxybiphenyl-1,2-dioxygenase from Rhodococcus rhodochrous

  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

A 2,3-dihydroxybiphenyl (2,3-DHBP) dioxygenase gene from a Rhodococcus sp. strain, named RrbphCI and involved in the degradation of polychlorinated biphenyls (PCBs), was synthesized. RrbphCI was expressed in Escherichia coli and its encoded enzyme was purified. SDS–PAGE analysis indicated that the size of the protein encoded by RrbphCI was about 32 kDa. The activity of the 2,3-DHBP dioxygenase was 82.8 U/mg when the substrate was 2,3-DHBP, with optimum pH 8.0 at 30°C, and optimum temperature was 40°C at pH 8.0. The RrbphCI gene was transformed into Pseudomonas putida strain EG11, to determine the ability of the enzyme to degrade 2,3-DHBP. The wild type EG11 degraded 61.86% of supplied 2,3-DHBP and the transformed EG11 (hosting the RrbphCI gene) utilized 52.68% after 2 min of treatment at 30°C. The overexpressed and purified enzyme was able to degrade 2,3-DHBP. The 2,3-DHBP dioxygenase is a key enzyme in the PCB degradation pathway. RrbphCI and its encoded 2,3-DHBP dioxygenase may have transgenic applications in bioremediation of PCBs.

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

References

  1. Furukawa K, Chakrabarty AM (1982) Involvement of plasmids in total degradation of chlorinated biphenyls. Appl Environ Microbiol 44:619–626

    PubMed  CAS  Google Scholar 

  2. Bedard DL, Haberl ML, May RJ, Brennan MJ (1987) Evidence for novel mechanisms of polychlorinated biphenyl metabolism in Alcaligenes eutrophus H850. Appl Environ Microbiol 53:1103–1112

    PubMed  CAS  Google Scholar 

  3. Mondello FJ (1989) Cloning and expression in Escherichia coli of Pseudomonas strain LB400 genes encoding polychlorinated biphenyl degradation. J Bacteriol 171:1725–1732

    PubMed  CAS  Google Scholar 

  4. Kimbara K, Hashimoto T, Fukuda M, Koana T, Takagi M, Oishi M, Yano K (1989) Cloning and sequencing of two tandem genes involved in degradation of 2, 3-dihydroxybiphenyl to benzoic acid in the polychlorinated biphenyl-degrading soil bacterium Pseudomonas sp. strain KKS102. J Bacteriol 171:2740–2747

    PubMed  CAS  Google Scholar 

  5. Ahmad D, Massé R, Sylvestre M (1990) Cloning and expression of genes involved in 4-chlorobiphenyl transformation by Pseudomonas testosteroni: homology to polychlorobiphenyl-degrading genes in other bacteria. Gene 86:53–61

    Article  PubMed  CAS  Google Scholar 

  6. De Mot R, Nagy I, De Schrijver A, Pattanapipitpaisal P, Schoofs G, Vanderleyden J (1997) Structural analysis of the 6 kb cryptic plasmid pFAJ2600 from Rhodococcus erythropolis NI86/21 and construction of Escherichia coli-Rhodococcus shuttle vectors. Microbiology 143:3137–3147

    Article  PubMed  Google Scholar 

  7. Maeda M, Chung SY, Song E, Kudo T (1995) Multiple genes encoding 2, 3-dihydroxybiphenyl 1, 2-dioxygenase in the Gram-positive polychlorinated biphenyl-degrading bacterium Rhodococcus erythropolis TA421, isolated from a termite ecosystem. Appl Environ Microbiol 61:549–555

    PubMed  CAS  Google Scholar 

  8. Masai E, Sugiyama K, Iwashita N, Shimizu S, Hauschild JE, Hatta T, Kimbara K, Yano K, Fukuda M (1997) The bphDEF meta-cleavage pathway genes involved in biphenyl/polychlorinated biphenyl degradation are located on a linear plasmid and separated from the initial bphACB genes in Rhodococcus sp. strain RHA1. Gene 187:141–149

    Article  PubMed  CAS  Google Scholar 

  9. Abramowicz DA (1995) Aerobic and anaerobic PCB biodegradation in the environment. Environ Health Perspect 103:97–99

    PubMed  CAS  Google Scholar 

  10. Abramowicz DA (1990) Aerobic and anaerobic biodegradation of PCBs: a review. Cri Rev Biotech 10:241–251

    Article  CAS  Google Scholar 

  11. Unterman R (1996) A history of PCB biodegradation. In: Crawford RL, Crawford DL (eds) Bioremediation: principles and applications. University Press, New York, Cambridge

    Google Scholar 

  12. Borja J, Taieon DM, Auresenia J, Gallardo S (2005) Polychlorinated biphenyls and their biodegradation. Process Biochem 40:1999–2013

    Article  CAS  Google Scholar 

  13. Pieper DH (2005) Aerobic degradation of polychlorinated biphenyls. Appl Microbiol Biotechnol 67:170–191

    Article  PubMed  CAS  Google Scholar 

  14. Pieper DH, Seeger M (2008) Bacterial metabolism of polychlorinated biphenyls. J Mol Microbiol Biotechnol 15:121–138

    PubMed  CAS  Google Scholar 

  15. Sakai M, Masai E, Asami H, Sugiyama K, Kimbara K, Fukuda M (2002) Diversity of 2, 3-dihydroxybiphenyl dioxygenase genes in a strong PCB degrader, Rhodococcus sp. strain. RHA1. J Biosci Bioeng 93:421–427

    PubMed  CAS  Google Scholar 

  16. Adebusoye SA, Picardal FW, Ilori MO, Amund OO, Fuqua C (2008) Characterization of multiple novel aerobic polychlorinated biphenyl (PCB)-utilizing bacterial strains indigenous to contaminated tropical African soils. Biodegradation 19:145–159

    Article  PubMed  CAS  Google Scholar 

  17. Finnerty WR (1992) The biology and genetics of the genus Rhodococcus. Annu Rev Microbiol 46:193–218

    Article  PubMed  CAS  Google Scholar 

  18. Rainey FA, Klatte S, Kroppenstedt RM, Stackebrandt E (1995) Dietzia, a new genus including Dietzia maris comb. nov., formerly Rhodococcus maris. Int J Syst Bacteriol 45:32–36

    Article  PubMed  CAS  Google Scholar 

  19. Kosono S, Maeda M, Fuji F, Arai H, Kudo T (1997) Three of the seven bphC genes of Rhodococcus erythropolis TA421, isolated from a termite ecosystem, are located on an indigenous plasmid associated with biphenyl degradation. Appl Environ Microbiol 63:3282–3285

    PubMed  CAS  Google Scholar 

  20. Maeda M, Roberts MS, Ohta Y, Fuji F, Travisano M, Kudo T (1998) Isolation and characterization of a new aromatic compound-degrading alkalitrophic bacteria. J Gen Appl Microbiol 44:101–106

    Article  PubMed  CAS  Google Scholar 

  21. Arai H, Kosono S, Taguchi K, Maeda M, Song E, Fuji F, Chang SY, Kudo T (1998) Two sets of biphenyl and PCB degradation genes on a linear plasmid in Rhodococcus erythropolis TA421. J Ferment Bioeng 86:595–599

    Article  CAS  Google Scholar 

  22. Sakai M, Masai E, Asami H, Sugiyama K, Kimbara K, Fukuda M (2002) Diversity of 2, 3-dihydroxybiphenyl dioxygenase genes in a strong PCB degrader, Rhodococcus sp. strain RHA1. J Biosci Bioeng 93:421–427

    PubMed  CAS  Google Scholar 

  23. Taguchi K, Motoyama M, Kudo T (2004) Multiplicity of 2, 3-dihydroxybiphenyl dioxygenase genes in the Gram-positive polychlorinated biphenyl degrading bacterium Rhodococcus rhodochrous K37. Biosci Biotechnol Biochem 68:787–795

    Article  PubMed  CAS  Google Scholar 

  24. Xiong AS, Yao QH, Peng RH, Li X, Fan HQ, Cheng ZM, Li Y (2004) A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequence. Nucl Acids Res 32:e98

    Article  PubMed  Google Scholar 

  25. Xiong AS, Yao QH, Peng RH, Li X, Fan HQ, Cheng ZM, Li Y (2006) PCR-based accurate synthesis of long DNA sequences. Nat Protoc 1:791–797

    Article  PubMed  CAS  Google Scholar 

  26. Peng RH, Xiong AS, Yao QH (2006) A direct and efficient PAGE-mediated overlap extension method for gene multiple-site mutagenesis. Appl Microbiol Biotechnol 73:234–240

    Article  PubMed  CAS  Google Scholar 

  27. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  28. Eltis LD, Hofmann B, Hecht HJ, Lünsdorf H, Timmis KN (1993) Purification and crystallization of 2, 3-dihydrobiphenyl 1, 2-dioxygenases. J Biol Chem 268:2727–2732

    PubMed  CAS  Google Scholar 

  29. Shuai JJ, Tian YS, Yao QH, Peng RH, Xiong F, Xiong AS (2010) Identification and analysis of polychlorinated biphenyls (PCBs)-biodegrading bacterial strains in Shanghai. Curr Microbiol 61(5):477–483. doi:10.1007/s00284-010-9641-2

    Article  PubMed  CAS  Google Scholar 

  30. Furukawa K, Arimura N (1987) Purification and properties of 2, 3-dihydroxybiphenyl dioxygenase from polychlorinated biphenl-degrading Pseudomonas pseudoalcaligenes and Pseudomonas aeruginosa carring the cloned bphC gene. J Bacteriol 169:924–927

    PubMed  CAS  Google Scholar 

  31. Drinker CK, Warren MF, Bennet GA (1937) The problem of possible systemic effects from certain chlorinated hydrocarbons. J Ind Hyg Toxicol 19:283–311

    CAS  Google Scholar 

  32. Rehmann L, Daugulis AJ (2008) Enhancement of PCB degradation by Burkholderia xenovorans LB400 in biphasic systems by manipulating culture conditions. Biotechnol Bioeng 99:521–528

    Article  PubMed  CAS  Google Scholar 

  33. Focht DD (1995) Strategies for the improvement of aerobic metabolism of polychlorinated biphenyls. Curr Opin Microbiol 6:341–346

    CAS  Google Scholar 

  34. Fava F, Bertin L (1999) Use of exogenous specialized bacteria in the biological detoxification of a dump site-polychlorinated biphenyl-contaminated soil in slurry phase conditions. Biotechnol Bioeng 64:240–249

    Article  PubMed  CAS  Google Scholar 

  35. Fava F, Di Gioia D, Marchetti L (2000) Role of the reactor configuration in the biological detoxification of a dump site polychlorobiphenyl-contaminated soil in lab slurry phase conditions. Appl Microbiol Biotechnol 53:243–248

    Article  PubMed  CAS  Google Scholar 

  36. Köller G, Moder M, Czihal K (2000) Peroxidative degradation of selected PCB: a mechanistic study. Chemosphere 41:1827–1834

    Article  PubMed  Google Scholar 

  37. Di Toro S, Zanaroli G, Fava F (2006) Intensification of the aerobic bioremediation of an actual site soil historically contaminated by polychlorinated biphenyls (PCBs) through bioagumentation with non acclimated, complex source of microorganisms. Microb Cell Fact 5:11

    Article  PubMed  Google Scholar 

  38. Asturias JA, Timmis KN (1993) Three different 2, 3-dihydroxybiphenyl-1, 2-dioxygenase genes in the Gram-positive polychlorobiphenyl-degrading bacterium Rhodococcus globerulus P6. J Bacteriol 175:4631–4640

    PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The research was supported by the Hi-Tech Research and Development 863 Program of China (2008AA10Z401).

Author information

Authors and Affiliations

  1. Shanghai Key Laboratory of Agricultural Genetics and Breeding, Biotechnological Research Institute, Shanghai Academy of Agricultural Sciences, 2901 Beidi Rd, 201106, Shanghai, China

    Fei Xiong, Jian-Jun Shuai, Ri-He Peng, Yong-Sheng Tian, Wei Zhao, Quan-Hong Yao & Ai-Sheng Xiong

  2. College of Bioscience and Biotechnology, Yangzhou University, 225009, Yangzhou, China

    Fei Xiong & Jian-Jun Shuai

Authors
  1. Fei Xiong
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jian-Jun Shuai
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ri-He Peng
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yong-Sheng Tian
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Wei Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Quan-Hong Yao
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ai-Sheng Xiong
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Quan-Hong Yao or Ai-Sheng Xiong.

Additional information

The authors Fei Xiong and Jian-Jun Shuai contributed equally to this article.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOC 695 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Xiong, F., Shuai, JJ., Peng, RH. et al. Expression, purification and functional characterization of a recombinant 2,3-dihydroxybiphenyl-1,2-dioxygenase from Rhodococcus rhodochrous . Mol Biol Rep 38, 4303–4308 (2011). https://doi.org/10.1007/s11033-010-0554-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-010-0554-8

Keywords

Search

Navigation