Applied Microbiology and Biotechnology

, Volume 97, Issue 6, pp 2483–2491 | Cite as

Cloning of a dibutyl phthalate hydrolase gene from Acinetobacter sp. strain M673 and functional analysis of its expression product in Escherichia coli

  • Jun WuEmail author
  • Xuewei Liao
  • Fangbo YuEmail author
  • Zhongbo Wei
  • Liuyan Yang
Biotechnologically relevant enzymes and proteins


A dibutyl phthalate (DBP) transforming bacterium, strain M673, was isolated and identified as Acinetobacter sp. This strain could not grow on dialkyl phthalates, including dimethyl, diethyl, dipropyl, dibutyl, dipentyl, dihexyl, di(2-ethylhexyl), di-n-octyl, and dinonyl phthalate, but suspensions of cells could transform these compounds to phthalate via corresponding monoalkyl phthalates. During growth in Luria–Bertani medium, M673 produced the high amounts of non-DBP-induced intracellular hydrolase in the stationary phase. One DBP hydrolase gene containing an open reading frame of 1,095 bp was screened from a genomic library, and its expression product hydrolyzed various dialkyl phthalates to the corresponding monoalkyl phthalates.


Dibutyl phthalate (DBP) Transformation Acinetobacter sp. DBP hydrolase gene 



This work was supported by the National Natural Science Foundation (Project nos. 21107047 and 31100087), the Fundamental Research Funds for Central Universities of The People’s Republic of China (Project nos. 1107021154 and 1118021114), and two grants (Project nos. Y3100018 and 2011C23065) from Zhejiang provincial government.


  1. Akita K, Naitou C, Maruyama K (2001) Purification and characterization of an esterase from Micrococcus sp. YGJ1 hydrolyzing phthalate esters. Biosci Biotechnol Biochem 65:1680–1683CrossRefGoogle Scholar
  2. Anthonsen HW, Baptista A, Drabløs F, Martel P, Petersen SB, Sebastião M, Vaz L (1995) Lipases and esterases: a review of their sequences, structure and evolution. Biotechnol Annu Rev 1:315–371CrossRefGoogle Scholar
  3. Batie CJ, LaHaie E, Ballou DP (1987) Purification and characterization of phthalate oxygenase and phthalate oxygenase reductase from Pseudomonas cepacia. J Biol Chem 262:1510–1518Google Scholar
  4. Cartwright CD, Owen SA, Thompson IP, Burns RG (2000) Biodegradation of diethyl phthalate in soil by a novel pathway. FEMS Microbiol Lett 186:27–34CrossRefGoogle Scholar
  5. Chang H-K, Zylstra GJ (1998) Novel organization of the genes for phthalate degradation from Burkholderia cepacia DB01. J Bacteriol 180:6529–6537Google Scholar
  6. Colborn T, vom Saal FS, Soto AM (1993) Developmental effects of endocrine-disrupting chemicals in wildlife and humans. Environ Health Perspect 101:378–384CrossRefGoogle Scholar
  7. David RM, Moore MR, Cifone MA, Finney DC, Guest D (1999) Chronic peroxisome proliferation and hepatomegaly associated with the hepatocellular tumorigenesis of di(2-ethylhexyl)phthalate and the effects of recovery. Toxicol Sci 50:195–205CrossRefGoogle Scholar
  8. Eaton RW (2001) Plasmid-encoded phthalate catabolic pathway in Arthrobacter keyseri 12B. J Bacteriol 183:3689–3703CrossRefGoogle Scholar
  9. Eaton RW, Ribbons DW (1982a) Metabolism of dibutylphthalate and phthalate by Micrococcus sp. strain 12B. J Bacteriol 151:48–57Google Scholar
  10. Eaton RW, Ribbons DW (1982b) Metabolism of dimethylphthalate by Micrococcus sp. strain 12B. J Bacteriol 151:465–467Google Scholar
  11. Eaton RW, Ribbons DW (1982c) The transformation of phthalaldehyde by phthalate-grown Micrococcus strain 12B. Arch Biochem Biophys 215:289–295CrossRefGoogle Scholar
  12. Eaton RW, Ribbons DW (1982d) Utilization of phthalate esters by Micrococci. Arch Microbiol 132:185–188CrossRefGoogle Scholar
  13. Engelhardt G, Wallnöfer PR (1978) Metabolism of di- and mono-n-butyl phthalate by soil bacteria. Appl Environ Microbiol 35:243–246Google Scholar
  14. Fukuda M, Shimizu S, Okita N, Seto M, Masai E (1998) Structural alteration of linear plasmids encoding the genes for polychlorinated biphenyl degradation in Rhodococcus strain RHA1. Antonie van Leeuwenhoek 74:169–173CrossRefGoogle Scholar
  15. Hara H, Stewart GR, Mohn WW (2010) Involvement of a novel ABC transporter and monoalkyl phthalate ester hydrolase in phthalate ester catabolism by Rhodococcus jostii RHA1. Appl Environ Microbiol 76:1516–1523CrossRefGoogle Scholar
  16. Hoang TT, Karkhoff-Schweizer RR, Kutchma AJ, Schweizer HP (1998) A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212:77–86CrossRefGoogle Scholar
  17. Huang PC, Tien CJ, Sun YM, Hsieh CY, Lee CC (2008) Occurrence of phthalates in sediment and biota: relationship to aquatic factors and the biota-sediment accumulation factor. Chemosphere 73:539–544CrossRefGoogle Scholar
  18. Huff JE, Kluwe WM (1984) Phthalate esters carcinogenicity in F344/N rats and B6C3F1 mice. Prog Clin Biol Res 141:137–154Google Scholar
  19. Jobling S, Reynolds T, White R, Parker MG, Sumpter JP (1995) A variety of environmentally persistent chemicals, including some phthalate plasticizers, are weakly estrogenic. Environ Health Perspect 103:582–587CrossRefGoogle Scholar
  20. Keyser P, Pujar RW, Eaton RW, Ribbons DW (1976) Biodegradation of phthalates and their esters by bacteria. Environ Health Perspect 18:159–166CrossRefGoogle Scholar
  21. Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop RMII, Peterson KM (1995) Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166:175–176CrossRefGoogle Scholar
  22. Maruyama K, Akita K, Naitou C, Yoshida M, Kitamura T (2005) Purification and characterization of an esterase hydrolyzing monoalkyl phthalates from Micrococcus sp. YGJ1. J Biochem 137:27–32CrossRefGoogle Scholar
  23. Miller SA, Dykes DD, Polesky HF (1988) A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16:1215CrossRefGoogle Scholar
  24. Nakazawa T, Hayashi E (1977) Phthalate metabolism in Pseudomonas testosteroni: accumulation of 4,5-dihydroxyphthalate by a mutant strain. J Bacteriol 131:42–48Google Scholar
  25. Neu HC, Heppel LA (1965) The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts. J Biol Chem 240:3685–3692Google Scholar
  26. Nishioka T, Iwata M, Imaoka T, Mutoh M, Egashira Y, Nishiyama T, Shin T, Fujii T (2006) A mono-2-ethylhexyl phthalate hydrolase from a Gordonia sp. that is able to dissimilate di-2-ethylhexyl phthalate. Appl Environ Microbiol 72:2394–2399CrossRefGoogle Scholar
  27. Nomura Y, Harashima S, Oshima Y (1989) A simple method for detection of enzyme activities involved in the initial step of phthalate degradation in microorganisms. J Ferment Bioeng 67:291–296CrossRefGoogle Scholar
  28. Piersma AH, Verhoef A, te Biesebeek J, Pieters MN, Slob W (2000) Developmental toxicity of butyl benzyl phthalate in the rat using a multiple dose study design. Reprod Toxicol 14:417–425CrossRefGoogle Scholar
  29. Ribbons DW, Evans WC (1960) Oxidative metabolism of phthalic acid by soil pseudomonads. Biochem J 76:310–317Google Scholar
  30. Ribbons DW, Keyser P, Kunz DA, Taylor BF, Eaton RW, Anderson BN (1984) Microbial degradation of phthalates. In: Gibson DT (ed) Microbial degradation of organic compounds. Marcel Dekker, New York, pp 371–397Google Scholar
  31. Sambrook J, Russell DW (2001) Molecular Cloning: A Laboratory Manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  32. Staples CA, Peterson DR, Parkerton TF, Adams WJ (1997) The environmental fate of phthalate esters: a literature review. Chemosphere 35:667–749CrossRefGoogle Scholar
  33. Vikelsoe J, Thomsen M, Carlsen L (2002) Phthalates and nonylphenols in profiles of differently dressed soils. Sci Total Environ 296:105–116CrossRefGoogle Scholar
  34. Wang F, Xia XH, Sha YJ (2008) Distribution of phthalic acid esters in Wuhan section of the Yangtze River, China. J Hazard Mater 154:317–324CrossRefGoogle Scholar
  35. Yuan SY, Liu C, Liao CS, Chang BV (2002) Occurrence and microbial degradation of phthalate esters in Taiwan river sediments. Chemosphere 49:1295–1299CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  1. 1.State Key Laboratory of Pollution Control and Resource ReuseSchool of the Environment, Nanjing UniversityNanjingPeople’s Republic of China
  2. 2.Center for Analysis and Testing, Nanjing Normal UniversityNanjingPeople’s Republic of China
  3. 3.Department of Environmental Biology, School of the EnvironmentNanjing UniversityNanjingPeople’s Republic of China
  4. 4.Department of Environmental Sciences, School of Environment and Resource SciencesZhejiang Agriculture and Forestry UniversityLinanPeople’s Republic of China

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