Skip to main content

Purification and Characterization of Catechol 1,2-Dioxygenase from Acinetobacter sp. Y64 Strain and Escherichia coli Transformants

The Protein Journal volume 34, pages 421–433 (2015)Cite this article

Abstract

This study intends to purify and characterize catechol 1,2-dioxygenase (C1,2O) of phenol-degrading Acinetobacter sp. Y64 and of E. coli transformant. Acinetobacter sp. Y64 was capable of degrading 1000 mg/L of phenol within 14 ± 2 h at 30 °C, 160 rpm and pH of 7. One C1,2O of 36 kDa was purified using ammonium sulphate precipitation and Hitrap QFF column chromatograph with 49 % recovery and a 10.6-fold increase in purity. Purified Y64 C1,2O had temperature and pH optimum at 37 °C and pH 7.7 respectively with the Michaelis constant of 17.53 µM and the maximal velocity of 1.95 U/mg, respectively. The presence of Fe3+ or Fe2+ enhanced the activity of Y64 C1,2O while other compounds such as Ca2+, and EDTA had an inhibitory effect. 80 % of C1,2O activity remained using 4-nitrocatechol as substrate while 2 % remained using 3-methylcatechol compared with that using catechol. Y64 catA gene encoding C1,2O was amplified using PCR cloned into pET22b vector and expressed in Escherichia coli BL21 DE3 (pLysS) after transformation. Purified and cloned Y64 C1,2O show no significant differences in the biochemical properties. The phylogenetic tree based on the protein sequences indicates that these C1,2Os possess a common ancestry.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

43,34 €

Price includes VAT (Finland)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Abbreviations

BH:

Bushnell Haas

bp:

Basepair

C1,2O:

Catechol 1,2-dioxygenase

C2,3O:

Catechol 2,3 dioxygenase

IPTG:

Isopropyl β-d-1-thiogalactopyranoside

K m :

Michaelis constant

PCR:

Polymerase chain reaction

QFF:

Q Sepharose® Fast Flow

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

V max :

The maximum rate of reaction

References

  1. Nadaf NH, Ghosh JS (2011) Purification and characterization of catechol 1,2 dioxygenase from Rhodococcus sp. NCIM 2891. Res J Environ Earth Sci 3(5):608–613

    CAS  Google Scholar 

  2. Yan J, Wen J, Jia X, Caiyin Q, Hu Z (2007) Mutation of Candida tropicalis by irradiation with a He–Ne laser to increase its ability to degrade phenol. Appl Environ Microbiol 73(1):226–231

    Article  Google Scholar 

  3. Curran KA, Leavitt JM, Karim AS, Alper HS (2013) Metabolic engineering of muconic acid production in Saccharomyces cerevisiae. Metab Eng 15:55–66

    Article  CAS  Google Scholar 

  4. Niu W, Draths KM, Frost JW (2002) Benzene-free synthesis of adipic acid. Biotechnol Prog 18:201–211

    Article  CAS  Google Scholar 

  5. Kaneko A, Ishii Y, Kirimura K (2011) High-yield production of cis, cis-muconic acid from catechol in aqueous solution by biocatalyst. Chem Lett 40:381–383

    Article  CAS  Google Scholar 

  6. Polen T, Spelberg M, Bott M (2013) Toward biotechnological production of adipic acid and precursors from biorenewables. J Biotechnol 167:75–84

    Article  CAS  Google Scholar 

  7. Lin J, Sharma V, Milase R, Mbhense N (2015) Simultaneous enhancement of phenolic compound degradations by Acinetobacter strain V2 via a step-wise continuous acclimation process. J Basic Microbiol. doi:10.1002/jobm.201500263.7

    Google Scholar 

  8. Pessione E, Giuffrida MG, Mazzoli R, Caposio P, Landolfo S, Conti A, Giunta C, Gribaudo G (2001) The catechol 1,2 dioxygenase system of Acinetobacter radioresistens: isoenzymes, inductors and gene localisation. Biological Chemistry 382(8):1253–1261

    Article  CAS  Google Scholar 

  9. Yang L, Nguyen DM, Jia S, Reid JS, Yu LE (2013) Impacts of biomass burning smoke on the distributions and concentrations of C2–C5 dicarboxylic acids and dicarboxylates in a tropical urban environment. Atmos Environ 78:211–218

    Article  CAS  Google Scholar 

  10. Pillar EA, Zhou R, Guzman MI (2015) Heterogeneous oxidation of catechol. J Phys Chem A 119:10349–10359

    Article  CAS  Google Scholar 

  11. Desyaterik Y, Sun Y, Shen X, Lee T, Wang X, Wang T, Collett JL (2013) Speciation of “brown” carbon in cloud water impacted by agricultural biomass burning in eastern China. J Geophys Res-Atmos 118:7389–7399

    Article  CAS  Google Scholar 

  12. Ali S, Fernandez-Lafuente R, Cowan DA (1998) Meta-pathway degradation of phenolics by thermophilic Bacilli. Enzyme Microb Technol 23:462–468

    Article  CAS  Google Scholar 

  13. Nakajima H, Ishida T, Tanaka H, Horiike K (2002) Accurate measurement of near-micromolar oxygen concentrations in aqueous solutions based on enzymatic extradiol cleavage of 4-chlorocatechol: applications to improved low-oxygen experimental systems and quantitative assessment of back diffusion of oxygen from the atmosphere. J Biochem 131:523–531

    Article  CAS  Google Scholar 

  14. Bushnell DL, Haas HF (1941) The utilization of certain hydrocarbons by microorganisms. Kans Agric Exp Stn 199:653–673

    Google Scholar 

  15. Klibanov AM, Alberti BN, Morris ED, Felsin LM (1980) Enzymatic removal of toxic phenols and anilines from wastewaters. J Appl Biochem 2:414–421

    CAS  Google Scholar 

  16. Briganti F, Pessione E, Giunta C, Scozzafava A (1997) Purification, biochemical properties and substrate specificity of a catechol 1,2-dioxygenase from a phenol degrading Acinetobacter radioresistens. FEBS Lett 416(1):61–64

    Article  CAS  Google Scholar 

  17. Bradford M (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  CAS  Google Scholar 

  18. Laemmli UK (1970) Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  Google Scholar 

  19. Tsai S-C, Li Y-K (2007) Purification and characterization of a catechol 1,2-dioxygenase from a phenol degrading Candida albicans TL3. Arch Microbiol 187:199–206

    Article  CAS  Google Scholar 

  20. Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132:365–386

    CAS  Google Scholar 

  21. Vázquez-Laslop N, Lee H, Hu R, Neyfakh AA (2001) Molecular sieve mechanism of selective release of cytoplasmic proteins by osmotically shocked Escherichia coli. J Bacteriol 183(8):2399–2404

    Article  Google Scholar 

  22. Guzik U, Hupert-Kocurek K, Sitnik M, Wojcieszyńska D (2013) High activity catechol 1,2-dioxygenase from Stenotrophomonas maltophilia strain KB2 as a useful tool in cis, cis-muconic acid production. Antonie Van Leeuwenhoek 103(6):1297–1307

    Article  CAS  Google Scholar 

  23. Briganti F, Pessione E, Giunta C, Mazzoli R, Scozzafava A (2000) Purification and catalytic properties of two catechol 1,2-dioxygenase isozymes from benzoate-grown cells of Acinetobacter radioresistens. J Protein Chem 19(8):709–716

    Article  CAS  Google Scholar 

  24. Caposio P, Pessione E, Giuffrida G, Conti A, Landolfo S, Giunta C, Gribaudo G (2002) Cloning and characterization of two catechol 1,2-dioxygenase genes from Acinetobacter radioresistens S13. Res Microbiol 153(2):69–74

    Article  CAS  Google Scholar 

  25. Suzuki K, Ichimura A, Ogawa N, Hasebe A, Miyashita K (2002) Differential expression of two catechol 1,2-dioxygenases in Burkholderia sp. strain TH2. J Bacteriol 184(20):5714–5722

    Article  CAS  Google Scholar 

  26. Miguez CB, Greer CW, Ingram JM (1993) Purification and properties of chlorocatechol 1,2-dioxygenase from Alcaligenes denitrifcans BRI 6011. Can J Microbiol 39:1–5

    Article  CAS  Google Scholar 

  27. Patel RN, Hou CT, Felix A, Lillard MO (1976) Catechol 1,2-dioxygenase from Acinetobacter calcoaceticus: purification and properties. J Bacteriol 127:536–544

    CAS  Google Scholar 

  28. Strachan PD, Freer AA, Fewson CA (1998) Purification and characterization of catechol 1,2-dioxygenase from Rhodococcus rhodochrous NCIMB 13259 and cloning and sequencing of its catA gene. Biochem J 333(3):741–747

    Article  CAS  Google Scholar 

  29. Saxena P, Thakur IS (2005) Purification and characterization of catechol 1,2-dioxygenase of Pseudomonas fluorescens for degradation of 4-chlorobenzoic acid. Indian J Biotechnol 4:134–138

    CAS  Google Scholar 

  30. Wang C, You S, Wang S (2006) Purification and characterization of a novel catechol 1,2-dioxygenase from Pseudomonas aeruginosa with benzoic acid as a carbon source. Process Biochem 41:1594–1601

    Article  CAS  Google Scholar 

  31. Guzik U, Gren I, Hupert-Kocurek K, Wojcieszyńska D (2011) Catechol 1,2-dioxygenase from the new aromatic compounds—degrading Pseudomonas putida strain N6. Int Biodeterior Biodegrad 65:504–512

    Article  CAS  Google Scholar 

  32. Aoki K, Konohana T, Shinke R, Nishira H (1984) Purification and characterization of catechol 1,2-dioxygenase from aniline-assimilating Rhodococcus erythropolis AN-13. Agric Biol Chem 48:2087–2095

    Article  CAS  Google Scholar 

  33. Silva AS, Jacques RJS, Andreazza R, Bento LFW, Roesch FM, Camargo FAO (2013) Properties of catechol 1,2-dioxygenase in the cell free extract and immobilized extract of Mycobacterium fortuitum. Braz J Microbiol 44(1):291–297

    Article  CAS  Google Scholar 

  34. Murakami S, Wang CL, Naito A, Shinke R, Aoki K (1998) Purification and characterization of four catechol 1,2-dioxygenase isozymes from the benzamide-assimilating bacterium Arthrobacter BA-5-17. Microbiol Res 153:163–171

    Article  CAS  Google Scholar 

  35. An H-R, Park H-J, Kim E-S (2001) Cloning and expression of thermophilic catechol 1,2-dioxygenase gene (catA) from Streptomyces setonii. FEMS Microbiol Lett 195(1):17–22

    Article  CAS  Google Scholar 

  36. Guo M, Qu YY, Zhou JT, Li A, Uddin MS (2009) Characterization of catechol 1,2 dioxygenase from cell extracts of Sphingomonas xenophaga QYY. Sci China Ser B Chem 52:615–620

    Google Scholar 

  37. Giedraityte G, Kalediene L (2009) Catechol 1,2-dioxygenase from a α-naphthol degrading thermophilic Geobacillus sp. strain: purification and properties. Cent Eur J Biol 4(1):68–73

    CAS  Google Scholar 

  38. Pakala SB, Gorla P, Pinjari AB, Krovidi RK, Baru R, Yanamandra M, Merrick M, Siddavattam D (2007) Biodegradation of methyl parathion and p-nitrophenol: evidence for the presence of a p-nitrophenol 2-hydroxylase in a Gram-negative Serratia sp. strain DS001. Appl Microbiol Biotechnol 73(6):1452–1462

    Article  CAS  Google Scholar 

  39. Pandeeti EVP, Siddavattam D (2011) Purification and characterization of catechol 1,2-dioxygenase from Acinetobacter sp. DS002 and cloning, sequencing of partial catA gene. Indian J Microbiol 51(3):312–318

    Article  CAS  Google Scholar 

  40. Varga JM, Neujahr Y (1970) Purification and properties of catechol 1,2-dioxygenase from Trichosporon cutaneum. Eur J Biochem 12:427–434

    Article  CAS  Google Scholar 

  41. Houghton JE, Shanley MS (1994) Catabolic potential of Pseudomonads: a regulatory perspective. Dioscorides Press, Portland, pp 11–32

    Google Scholar 

  42. Sanakis Y, Mamma D, Christakopoulos P, Stamatis H (2003) Catechol 1,2-dioxygenase from Pseudomonas putida in organic media—an electron paramagnetic resonance study. Int J Biol Macromol 33(1–3):101–106

    Article  CAS  Google Scholar 

  43. Miyazawa D, Mukerjee-Dhar G, Shimura M, Hatta T, Kimbara K (2004) Genes for Mn(II)-dependent NahC and Fe(II)-dependent NahH located in close proximity in the thermophilic naphthalene and PCB degrader, Bacillus sp. JF8: cloning and characterization. Microbiology 150(4):993–1004

    Article  CAS  Google Scholar 

  44. Camargo FAO, Andreazza R, Baldoni DB, Bento FM (2012) Enzymatic activity of catechol 1,2-dioxygenase and catechol 2,3-dioxygenase produced by Gordonia polyisoprenivorans. Quim Nova 35(8):1587–1592

    Article  Google Scholar 

  45. Vetting MW, Ohlendorf DH (2000) The 1.8 Å crystal structure of catechol 1,2 dioxygenase reveals a novel hydrophobic helical zipper as a subunit linker. Structure 8:429–440

    Article  CAS  Google Scholar 

  46. Neidle EL, Hartnett C, Bonitz S, Ornston LN (1988) DNA sequence of the Acinetobacter calcoaceticus catechol 1,2 dioxygenase I structural gene cat A: evidence for evolutionary divergence of intradiol dioxigenases by acquisition of DNA sequence repetitions. J Bacteriol 170:4874–4880

    CAS  Google Scholar 

Download references

Author information

Affiliations

  1. Discipline of Microbiology, School of Life sciences, University of KwaZulu-Natal, Westville Campus, Private Bag X54001, Durban, 4001, South Africa

    J. Lin & R. N. Milase

Authors
  1. J. Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. R. N. Milase
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to J. Lin.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Lin, J., Milase, R.N. Purification and Characterization of Catechol 1,2-Dioxygenase from Acinetobacter sp. Y64 Strain and Escherichia coli Transformants. Protein J 34, 421–433 (2015). https://doi.org/10.1007/s10930-015-9637-7

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10930-015-9637-7

Keywords

  • Catechol 1,2 dioxygenase
  • Acinetobacter sp.
  • Enzyme purification
  • Gene cloning
  • Characterization