Rendiconti Lincei

, Volume 28, Issue 1, pp 1–9 | Cite as

Meta-cleavage pathway of phenol degradation by Acinetobacter sp. strain AQ5NOL 1

  • Siti Aqlima Ahmad
  • Nor Aripin Shamaan
  • Mohd Arif Syed
  • Ariff Khalid
  • Nor Arina Ab Rahman
  • Khalilah Abdul Khalil
  • Farrah Aini Dahalan
  • Mohd Yunus Shukor


The characterization of bacterial enzymatic pathways of phenol metabolism is important to better understand phenol biodegradation. Phenol hydroxylase is the first enzyme involved in the oxidative metabolism of phenol, followed by further degradation via either meta- or ortho-pathways. In this study, the first known instance of phenol degradation via the meta-pathway by a member of the genus Acinetobacter (Acinetobacter sp. strain AQ5NOL 1) is reported. Phenol hydroxylase converts phenol to catechol, which is then converted via the meta-pathway to 2-hydroxymuconic semialdehyde by the catechol 2,3-dioxygenase enzyme. Phenol hydroxylase extracted from strain AQ5NOL 1 was fully purified using DEAE-Sepharose®, DEAE-Sephadex®, Q-Sepharose® and Zorbax® Bioseries GF-250 gel filtration and was demonstrated by SDS-PAGE to have a molecular weight of 50 kDa. The phenol hydroxylase was purified to about 210.51 fold. The optimum pH and temperature for enzyme activities are 20 °C and 7–7.5, respectively. The apparent K m and V max values of phenol hydroxylase with phenol as the substrate were 13.4 µM and 2.5 µmol min−1 mg−1, respectively. The enzyme was stable at −20 °C for 36 days.


Purification Characterization Phenol hydroxylase Acinetobacter sp. 



This work was supported by the Research Grant Scheme (RUGS) 2009, Universiti Putra Malaysia (Vote No. 91851).


  1. Adams D, Ribbons DW (1998) The metabolism of aromatic ring fission products by baciluus stearothermophilus IC3. J Gen Microbiol 134:3179–3185Google Scholar
  2. Ahmad SA, Shamaan NA, Arif NM, Shukor MYA, Syed MA (2011) Identification and characterization of a phenol degrading Acinetobacter sp. strain AQ5NOL 1. Aust J Basic Appl Sci 5:1035–1045Google Scholar
  3. Ahmad SA, Shamaan NA, Arif NM, Koon GB, Shukor MYA, Syed MA (2012) Enhancement of biodegradation of phenol by immobilized cells of Acinetobacter sp. strain AQ5NOL 1. World J Microbiol Biotech 28:347–352CrossRefGoogle Scholar
  4. Ahmad SA, Shukor MY, Shamaan NA, Rahman NAA, Dahalan FA, Khalil KA, Syed MA (2015) Effects of pesticides and respiratory inhibitors on phenol degradation by Acinetobacter sp. strain AQ5NOL 1 immobilized in gellan gum. J Pure Appl Microbiol 9:489–495Google Scholar
  5. Arif NM, Ahmad SA, Syed MA, Shukor MY (2013) Isolation and characterization of a phenol-degrading Rhodococcus sp. strain AQ5NOL 2 KCTC 11961BP. J Basic Microbiol 53:9–19CrossRefGoogle Scholar
  6. Basha KM, Rajendran A, Thangavelu V (2010) Recent advances in the biodegradation of phenol: a review. Asian J Exp Biol Sci 1:219–234Google Scholar
  7. 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 72:248–252CrossRefGoogle Scholar
  8. Briganti F, Pessione E, Giunta C, Scozzafava A (1997) Purification, biochemical properties and substrate specificity of a catechol 1,2-dioxygenase from phenol degrading Acinetobacter radioresistens. FEBS Lett 416:61–64CrossRefGoogle Scholar
  9. Carvalho MB, Martins I, Leitão MC, Garcia H, Rodrigues C, San Romão V, McLellan I, Hursthouse A, Silva Pereira C (2009) Screening pentachlorophenol degradation ability by environmental fungal strains belonging to the phyla Ascomycota and Zygomycota. J Ind Microbiol Biotechnol 36:1249–1256CrossRefGoogle Scholar
  10. Cassidy MB, Mullineers H, Lee H, Trevors JT (1997) Mineralization of pentachlorophenol in a contaminated soil by Pseudomonassp UG30 cells encapsulated in κ-carrageenan. J Ind Microbiol Biotechnol 19:43–48CrossRefGoogle Scholar
  11. Chakraborty S, Bhattacharya T, Patel TN, Tiwari KK (2010) Biodegradation of phenol by native microorganisms isolated from coke processing wastewater. J Environ Biol 31:293–296Google Scholar
  12. Divari S, Valetti F, Caposia P, Pessione E, Cavaletto M, Griva E, Gribaudo G, Gilardi G, Giunta G (2003) The oxygenase component of phenol hydroxylase from Acinetobacter radioresistens S13. Eur J Biochem 270:2244–2253CrossRefGoogle Scholar
  13. Dong X, Hong Q, He L, Jiang X, Li S (2008) Characterization of phenol-degrading bacterial strains isolated from natural soil. Int Biodeterior Biodeg 62:257–262CrossRefGoogle Scholar
  14. El-Haleem D, Beshay U, Abdelhamid A, Moawad H, Zaki S (2003) Effect of mixed nitroghen sources on biodegradtion of phenol by immobilized Acinetobacter: sp strain W-17. Afr J Biotechnol 2:8–12CrossRefGoogle Scholar
  15. Etoumi A, El Musrati I, El Gammoudi B, El Behlil M (2008) The reduction of wax precipitation in waxy crude oils by Pseudomonas species. J Ind Microbiol Biotechnol 35:1241–1245CrossRefGoogle Scholar
  16. Farrell A, Quilty B (2002) Substrate-dependent autoaggregation of Pseudomonas putida CP1 during the degradation of mono-chlorophenols and phenol. J Ind Microbiol Biotechnol 28:316–324CrossRefGoogle Scholar
  17. González G, Herrera G, García T, Peña M (2001) Biodegradation of phenolic industrial wastewater in a fluidized bed bioreactor with immobilized cells of Pseudomonas putida. Biores Technol 80:137–142CrossRefGoogle Scholar
  18. Hannaford AM, Kuek C (1999) Aerobic batch degradation of phenol using immobilized Pseudomonas putida. J Ind Microbiol Biotechnol 22:121–126CrossRefGoogle Scholar
  19. Hao OJ, Kim MH, Seagren EA, Kim H (2002) Kinetics of phenol and chlorophenol utilization by Acinetobacter species. Chemosphere 46:797–807CrossRefGoogle Scholar
  20. Hori TSF, Avilez IM, Inoue LK, Moraes G (2006) Metabolical changes induced by chronic phenol exposure in matrinxã Brycon cephalus (Teleostei: Characidae) juveniles. Comp Biochem Physiol Part C 143:67–72CrossRefGoogle Scholar
  21. Jiang HL, Tay JH, Tay STL (2004) Changes in structure, activity and metabolism of erobic granules as a microbial response to high phenol loading. Appl Microbiol Biotechnol 63:602–608CrossRefGoogle Scholar
  22. Jiang HL, Tay STL, Maszenan AM, Tay JH (2006) Physiol traits bacterial strains isolated phenol degrading aerobic granules. FEMS Microbiol Ecol 57:182–191CrossRefGoogle Scholar
  23. Kagle J, Hay AG (2006) Phenylacetylene reversibly inhibits the phenol hydroxylase of Pseudomonas sp. CF600 at high concentrations but is oxidized at lower concentrations. Appl Microbiol Biotechnol 72:306–315CrossRefGoogle Scholar
  24. Kirchner U, Westphal AH, Muller R, Berkel WJHV (2003) Phenol hydroxylase from Bacillus thermoglucosidasius A7, a two-protein component monooxygenase with a dual roel for FAD. J Biol Chem 278:47545–47553CrossRefGoogle Scholar
  25. Muller RH, Babel W (1996) Growth rate-dependent expression of phenolassimilation pathways in Alcaligenes eutrophus JMP 134- the influence of formate as an auxiliary energy sources on phenol conversion characteristics. Appl Microbiol Biotechnol 46:156–162CrossRefGoogle Scholar
  26. Nawawi NM, Ahmad SA, Shukor MY, Syed MA, Khalil KA, Ab Rahman NA, Dahalan FA, Ibrahim AL (2016) Statistical optimization for improvement of phenol degradation by Rhodococcus sp. NAM 81. J Envion Biol 37:443–451Google Scholar
  27. Norazah MN, Jayasree N, Ahmad SA, Shukor MY, Abdul Latif I (2015) Disrupting Rhodococcus sp: a competent method for genomics and proteomics. J Chem Pharm Sci 8:336–341Google Scholar
  28. Paller G, Hommel RK, Kleber HP (1995) Phenol degradation by Acinetobacter calcoaceticus NCIB 8250. J Basic Microbiol 35:325–335CrossRefGoogle Scholar
  29. Saa L, Jaureguibeitia A, Largo E, Llama MJ, Serra JL (2009) Cloning, purification and characterization of two components of phenol hydroxylase from Rhodococcus erythropolis UPV-1. Appl Microbiol Biotechnol 86:201–211CrossRefGoogle Scholar
  30. Salleh AB, Ghazali FM, Rahman RNZA, Basri M (2003) Bioremediation of petroleum hydrocarbon pollution. Afr J Biotechnol 2:411–425Google Scholar
  31. Scopes RK (1998) Protein purification, principles and practice. Springer-Verlag, New YorkGoogle Scholar
  32. Sharma A, Thakur IS, Dureja P (2009) Enrichment, isolation and characterization of pentachlorophenol degrading bacterium Acinetobacter sp. ISTPCP-3 from effluent discharge site. Biodegradation 20:643–650CrossRefGoogle Scholar
  33. Straube G (1987) Phenol hydroxylase from Rhodococcus sp. P1. J Basic Microbiol 27:229–232CrossRefGoogle Scholar
  34. Suhaila YN, Rosfarizan M, Ahmad SA, Latif IA, Ariff AB (2013) Nutrients and culture conditions requirements for the degradation of phenol by Rhodococcus UKMP-5M. J Environ Biol 34:635–643Google Scholar
  35. Tsai SC, Tsai LD, Li YK (2005) An isolated candida albicans TL3 capable of degrading phenol at large concentration. Biosci Biotechnol Biochem 69:2358–2367CrossRefGoogle Scholar
  36. Tuan NN, Hsieh HC, Lin YW, Huang SY (2011) Analysis of bacterial degradation pathways for long-chain alkylphenols involving phenol hydroxylase, alkylphenol monooxygenase and catechol dioxygenase genes. Biores Technol 102:4232–4240CrossRefGoogle Scholar
  37. van Schie PM, Young LY (2000) Biodegradation of phenol mechanisms and applications. Bioremed J 4:1–18CrossRefGoogle Scholar
  38. Veenagayathri K, Vasudevan N (2011) Ortho and mete cleavage dioxygenase detected during the degradation of phenolic compounds by a moderately halophilic bacterial consortium. Int Res J Microbiol 2:406–414Google Scholar
  39. Viggor S, Heinaru E, Kuennapas A, Heinaru A (2008) Evaluation of different phenol hydroxylase-possessing phenol-degrading pseudomonas by kinetic parameters. Biodegradation 19:759–769CrossRefGoogle Scholar
  40. Wang Y, Tian Y, Han B, Zhao HB, Bi JN, Cai BL (2007) Biodegradation of phenol by free and immobilized Acinetobacter sp. strain PD12. J Environ Sci 19:222–225CrossRefGoogle Scholar
  41. Yang CF, Lee CM (2007) Enrichment, isolation, and characterization of phenol-degrading Pseudomonas resinovorans strain P-1 and Brevibacillus sp. strain P-6. Int Biodeterior Biodeg 59:206–210CrossRefGoogle Scholar
  42. Yemendzhiev H, Gerginova M, Krastanov A, Stoilova I, Alexieva Z (2008) Growth of Trametes versicolor on phenol. J Ind Microbiol Biotechnol 35:1309–1312CrossRefGoogle Scholar
  43. Yotova L, Tzibranska I, Tileva F, Markx GH, Georgieva N (2009) Kinetics of the biodegradation of phenol in wastewaters from the chemical industry by covalently immobilized Trichosporon cutaneum cells. J Ind Microbiol Biotechnol 36:367–372CrossRefGoogle Scholar
  44. Zaki S (2006) Detection of meta- and ortho-cleavage dioxygenases in bacterial phenol-degraders. J Appl Sci Environ Manag 10:75–81Google Scholar
  45. Zhu C, Zhang L, Zhao L (2008) Molecular cloning, genetic organization of gene cluster encoding phenol hydroxylase and catechol 2,3-dioxygenase in Alcaligenes faecalis IS-46. World J Microbiol Biotechnol 24:1687–1695CrossRefGoogle Scholar

Copyright information

© Accademia Nazionale dei Lincei 2016

Authors and Affiliations

  • Siti Aqlima Ahmad
    • 1
  • Nor Aripin Shamaan
    • 2
  • Mohd Arif Syed
    • 1
  • Ariff Khalid
    • 3
  • Nor Arina Ab Rahman
    • 1
  • Khalilah Abdul Khalil
    • 4
  • Farrah Aini Dahalan
    • 5
  • Mohd Yunus Shukor
    • 1
  1. 1.Department of Biochemistry, Faculty of Biotechnology and Biomolecular SciencesUniversiti Putra MalaysiaSerdangMalaysia
  2. 2.Faculty of Medicine and Health SciencesUniversiti Sains Islam MalaysiaKuala LumpurMalaysia
  3. 3.Faculty of Health SciencesUniversiti Kebangsaan MalaysiaKuala LumpurMalaysia
  4. 4.The School of Environmental EngineeringUniversiti Malaysia PerlisArauMalaysia
  5. 5.Biomolecular Science Program, School of Biology, Faculty of Applied SciencesUniversiti Teknologi MARAShah AlamMalaysia

Personalised recommendations