Skip to main content
SpringerLink
Log in

Cloning, sequencing, and expression of a novel epoxide hydrolase gene from Rhodococcus opacus in Escherichia coli and characterization of enzyme

  • Biotechnologically Relevant Enzymes and Proteins
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

An epoxide hydrolase gene of about 0.8 kb was cloned from Rhodococcus opacus ML-0004, and the open reading frame (ORF) sequence predicted a protein of 253 amino acids with a molecular mass of about 28 kDa. An expression plasmid carrying the gene under the control of the tac promotor was introduced into Escherichia coli, and the epoxide hydrolase gene was successfully expressed in the recombinant strains. Some characteristics of purified recombinant epoxide hydrolase were also studied. Epoxide hydrolase showed a high stereospecificity for l(+)-tartaric acid, but not for d(+)-tartaric acid. The epoxide hydrolase activity could be assayed at the pH ranging from 3.5 to 10.0, and its maximum activity was obtained between pH 7.0 and 7.5. The enzyme was sensitive to heat, decreasing slowly between 30°C and 40°C, and significantly at 45°C. The enzyme activity was activated by Ca2+ and Fe2+, while strongly inhibited by Ag+ and Hg+, and slightly inhibited by Cu2+, Zn2+, Ba2+, Ni+, EDTA–Na2 and fumarate.

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.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Arand M, Hemmer H, Durk H, Baratti J, Archelas A, Furstoss R, Oesch F (1999) Cloning and molecular characterization of a soluble epoxide hydrolase from Aspergillus niger that is related to mammalian microsomal epoxide hydrolase. Biochem J 344:273–280

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Archelas A, Furstoss R (2001) Synthetic applications of epoxide hydrolases. Curr Opin Chem Biol 5:112–119

    Article  CAS  PubMed  Google Scholar 

  • Blée E, Schuber F (1995) Stereocontrolled hydrolysis of the linoleic acid monoepoxide regioisomers catalyzed by soybean epoxide hydrolase. Eur J Biochem 230:229–234

    Article  PubMed  Google Scholar 

  • Botes AL (1999) Affinity purification and characterization of a yeast epoxide hydrolase. Biotechnol Lett 21:511–517

    Article  CAS  Google Scholar 

  • 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

    CAS  PubMed  Google Scholar 

  • Buch ML, Montgomery R, Porter WL (1952) Identification of organic acids on paper chromatograms. Anal Chem 24:489–491

    Article  CAS  Google Scholar 

  • Bučko M, Vikartovska A, Lacik I, Kollarikova G, Gemeiner P, Patoprsty V, Brygin M (2005) Immobilization of a whole-cell epoxide-hydrolyzing biocatalyst in sodium alginate-cellulose sulfate-poly(methylene-co-guanidine) capsules using a controlled encapsulation process. Enzyme Microb Technol 36:118–126

    Article  Google Scholar 

  • Chung CT, Niemela SL, Miller RH (1989) One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc Natl Acad Sci USA 86:2172–2175

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Faber K, Mischitz M, Kroutil W (1996) Microbial epoxide hydrolases. Acta Chem Scand 50:249–258

    Article  CAS  Google Scholar 

  • Frederich MA, Roger B, Robert EK, David DM, Seidman JG, John AS, Kevin S (1999) Short protocols in molecular biology, 4th edn. Wiley pp2–12. ISBN: 0471250929

  • Grogan G, Roberts SM, Willetts AJ (1996) Novel aliphatic epoxide hydrolase activities from dematiaceous fungi. FEMS Microbiol Lett 141:239–243

    Article  CAS  PubMed  Google Scholar 

  • Hammock BD, Grant DD, Storms DH (1997) In: Sipes I, McQueen C, Gandolfi A (eds) Comprehensive toxicology, ch. 18.3, Pergamon, Oxford

    Google Scholar 

  • Jaeger KE, Eggert T, Eipper A, Reetz MT (2001) Directed evolution and the creation of enantioselective biocatalysts. Appl Microbiol Biotechnol 55:519–530

    Article  CAS  PubMed  Google Scholar 

  • Kamatani Y, Ogino K (1977) Production of l(+)-tartaric acid. US Patent 4,011,135

  • Kolb HC, Van Nieuwenhze MS, Sharpless KB (1994) For an alternative approach to the synthesis of optically active 1,2-diols. Chem Rev 94:2483–2547

    Article  CAS  Google Scholar 

  • Kroutil W, Genzel Y, Pietzsch M, Syldatk C, Faber K (1998) Purification and characterization of a highly selective epoxide hydrolase from Nocardia sp. EH1. J Biotechnol 61:143–150

    Article  CAS  PubMed  Google Scholar 

  • Kurillová L’, Gemeiner P, Vikartovská A, Miková H, Rosenberg M, Ilavský M (2000) Calcium pectate gel beads for cell entrapment. 6. Morphology of stabilized and hardened calcium pectate gel beads with cells for immobilized biotechnology. J Microencapsul 17:279–296

    Article  PubMed  Google Scholar 

  • Labuschagne M, Botes AL, Albertyn J (2004) Cloning and sequencing of an epoxide hydrolase gene from Rhodosporidium paludigenum. DNA Seq 15:202–205

    Article  CAS  PubMed  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of bacteriphoge T4. Nature 227:680–685

    Article  CAS  PubMed  Google Scholar 

  • Linderman RJ, Walker EA, Haney C, Roe RM (1995) Determination of the regiochemistry of insect epoxide hydrolase catalyzed epoxide hydration of juvenile hormone by 18O-labelling studies. Tetrahedron 51:10845–10856

    Article  CAS  Google Scholar 

  • Mischitz M, Kroutil W, Wandel U, Faber K (1995) Asymmetric microbial hydrolysis of epoxides. Tetrahedron: Asymmetry 6:1261–1272

    Article  CAS  Google Scholar 

  • Miura Y, Yutani K, Takesue H, Fujii K (1977) Microbiological process for preparing L-tartaric acid in presence of surfactants. US Patent 4,017,362

  • Morisseau C, Archelas A, Guitton C, Faucher D, Furstoss P, Baratti JC (1999) Purification and characterization of a highly enantioselective epoxide hydrolase from Aspergillus niger. Eur J Biochem 263:386–395

    Article  CAS  PubMed  Google Scholar 

  • Osprian I, Kroutil W, Mischitz M, Faber K (1997) Biocatalytic resolution of 2-methyl-2-(aryl)alkyloxiranes using novel bacterial epoxide hydrolases. Tetrahedron: Asymmetry 8:65–71

    Article  CAS  Google Scholar 

  • Rosenberg M, Miková H, Krištofíkova L’ (1999) Production of L-tartaric acid by immobilized bacterial cells Nocardia tartaricans. Biotechnol Lett 21:491–495

    Article  CAS  Google Scholar 

  • Rui LY, Cao L, Chen W, Reardon KF, Wood TK (2005) Protein engineering of epoxide hydrolase from Agrobacterium radiobacter AD1 for enhanced activity and enantioselective production of (R)-1-phenylethane-1,2-diol. Appl Environ Microb 71:3995–4003

    Article  CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (2001) Molecular cloning: a laboratory manual, vol. 1, 3rd edn. Cold Spring Harbor Lab. Press, NY, pp 26–30. ISBN 7-03-010338-6

    Google Scholar 

  • Steinreiber A, Faber K (2001) Microbial epoxide hydroxylases for preparative biotransformations. Curr Opin Biotechnol 12:552–558

    Article  CAS  PubMed  Google Scholar 

  • van Loo B, Spelberg JHL, Kingma J, Sonke T, Wubbolts MG, Janssen DB (2004) Directed evolution of epoxide hydrolase from A-radiobacter toward higher enantioselectivity by error-prone PCR and DNA shuffling. Chem Biol 11:981–990

    Article  PubMed  Google Scholar 

  • Visser H, De Bont JAM, Verdoes JC (1999) Isolation and characterization of the epoxide hydrolase-encoding gene from Xanthophyllomyces dendrorhous. Appl Microbiol Biotechnol 65:5459–5463

    CAS  Google Scholar 

  • Visser H, Vreugdenhil S, de Bont JAM, Verdoes JC (2000) Cloning and characterization of an epoxide hydrolase-encoding gene from Rhodotorula glutinis. Appl Microbiol Biotechnol 53:415–419

    Article  CAS  PubMed  Google Scholar 

  • Weijers CAGM, de Bont JAM (1999) Epoxide hydrolases from yeasts and other sources: versatile tools in biocatalysis. J Mol Catal B 6:199–214

    Article  CAS  Google Scholar 

  • Zhang JG, Qian YJ (2000) Production of l(+)-tartaric acid by immobilized Corynebacterium sp. JZ-1. Chin J Biotechnol 16:188–192

    Google Scholar 

  • Zhao LS, Han B, Huang ZL, Miller M, Huang HJ, Malashock DS, Zhu ZL, Milan A, Robertson DE, Weiner DP, Burk MJ (2004) Epoxide hydrolase-catalyzed enantioselective synthesis of chiral 1,2-diols via desymmetrization of meso-epoxides. J Am Chem Soc 126:11156–11157

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgement

We wish to thank Dr. Fengjie Cui, School of Food Engineering and Biotechnology, Jiangsu University for his thoughtful suggestions and correction of the English version on an earlier manuscript.

Author information

Authors and Affiliations

  1. Institute of Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, People’s Republic of China

    Zhiqiang Liu & Yuguo Zheng

  2. Department of Plant Science, North Dakota State University, Fargo, ND, 58105, USA

    Yin Li & Yingying Xu

  3. Institute of Quality and Standard for Agricultural Products, Zhejiang Academy of Agricultural Sciences, Hangzhou, 310021, People’s Republic of China

    Lifeng Ping

Authors
  1. Zhiqiang Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yin Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yingying Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lifeng Ping
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yuguo Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yuguo Zheng.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Liu, Z., Li, Y., Xu, Y. et al. Cloning, sequencing, and expression of a novel epoxide hydrolase gene from Rhodococcus opacus in Escherichia coli and characterization of enzyme. Appl Microbiol Biotechnol 74, 99–106 (2007). https://doi.org/10.1007/s00253-006-0635-8

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-006-0635-8

Keywords

Search

Navigation