Biodegradation

, Volume 23, Issue 4, pp 561–573 | Cite as

Growth of Acinetobacter gerneri P7 on polyurethane and the purification and characterization of a polyurethanase enzyme

  • Gary T. Howard
  • William N. Norton
  • Timothy Burks
Original Article

Abstract

A soil microorganism, designated as P7, was characterized and investigated for its ability to degrade polyurethane (PU). This bacterial isolate was identified as Acinetobacter gerneri on the basis of 16 s rRNA sequencing and biochemical phenotype analysis. The ability of this organism to degrade polyurethane was characterized by the measurement of growth, SEM observation, measurement of electrophoretic mobility and the purification and characterization of a polyurethane degrading enzyme. The purified protein has a molecular weight of approximately 66 kDa as determined by SDS-PAGE. Substrate specificity was examined using p-nitrophenyl substrates with varying carbon lengths. The highest substrate specificity was observed using p-nitrophenyl-propanate with an activity of 37.58 ± 0.21 U mg−1. Additionally, the enzyme is inhibited by phenylmethylsulfonylfluoride and by ethylenediamine-tetra acetic acid. When grown on Impranil DLN YES medium, a lag phase was noted for the first 3 h which was followed by logarithmic growth for 5 h. For the linear portion of growth between 5 and 9 h, a μ value of 0.413 doublings h−1 was calculated. After 9 h of incubation the cell number dramatically decreased resulting in a chalky precipitate. Measurements of electrophoretic mobility indicated the formation of a complex between the PU and A. gerneri P7 cells. A hybrid zeta potential had been generated between the cells and polyurethane. Further evidence for a complex was provided by SEM observation where cells appeared to cluster along the surface of polyurethane particles and along edges of polyurethane films. Occasionally, the cells established an anchor-like structure that connected the cells to polyurethane particles.

Keywords

Polyurethanase Acinetobacter Biodegradation 

References

  1. Akutsu Y, Nakajima-Kambe T, Nomura N, Nakahara T (1998) Purification and properties of a polyester polyurethane-degrading enzyme from Comamonas acidovorans TB-35. Appl Environ Microbiol 64:62–67PubMedGoogle Scholar
  2. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402PubMedCrossRefGoogle Scholar
  3. Baumann P (1968) Isolation of Acinetobacter from soil and water. J Bacteriol 96:39–42PubMedGoogle Scholar
  4. Bayer O (1947) Polyurethanes. Mod Plast 24:149–152Google Scholar
  5. Bergogne-Bérézin E, Towner KJ (1996) Acinetobacter spp. as nosocomial pathogens: microbiological, clinical, and epidemiological features. Clin Microbiol Rev 9:148–165PubMedGoogle Scholar
  6. Berlau J, Aucken HM, Houang E, Pitt TL (1999) Isolation of Acinetobacter spp. including A. baumannii from vegetables: implications for hospital-acquired infections. J Hosp Infect 42:201–204PubMedCrossRefGoogle Scholar
  7. Bernards AT, de Beaufort AT, Dijkshoorn L, van Boven CPA (1997) Outbreak of septicaemia in neonates caused by Acinetobacter junii investigated by amplified ribosomal DNA restriction analysis (ARDRA) and four typing methods. J Hosp Infect 35:129–140PubMedCrossRefGoogle Scholar
  8. Blake RC, Howard GT (1998) Adhesion and growth of a Bacillus sp. on a polyesterurethane. Int Biodeterio Biodegrad 42:63–73CrossRefGoogle Scholar
  9. Bouvet PJM, Grimont PAD (1986) Taxonomy of the genus Acinetobacter with the recognition of Acinetobacter baumannii sp. nov. Acinetobacter haemolyticus sp. nov. Acinetobacter johnsonii sp. nov. and Acinetobacter junii sp. nov. and emended descriptions of Acinetobacter calcoaceticus and Acinetobacter lwofii. Int J Syst Bacteriol 36:228–240CrossRefGoogle Scholar
  10. 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 72:248–254PubMedCrossRefGoogle Scholar
  11. Carr EL, Kampfer P, Patel BKC, Gurtler V, Seviour RJ (2003) Seven novel species of Acinetobacter isolated from activated sludge. Int J Syst Evol Microbiol 53:953–963PubMedCrossRefGoogle Scholar
  12. Crabbe JR, Campbell JR, Thompson L, Walz SL, Schultz WW (1994) Biodegradation of a collodial ester-based polyurethane by soil fungi. Int Biodeterio Biodegrad 33:103–113CrossRefGoogle Scholar
  13. DeSantis TZ, Hugenholtz P, Keller K, Brodie EL, Larsen N, Piceno YM, Phan R (2006) A server for comparative analysis of 16 s rRNA genes. Nucleic Acids Res 34:394–399CrossRefGoogle Scholar
  14. Deshpande MV, Eriksson KE, Peterson LG (1984) An assay for selective determination of exo-1,4-β-glucanases in a mixture of cellulolytic enzymes. Anal Biochem 138:481–487PubMedCrossRefGoogle Scholar
  15. El-Sayed AHMM, Mahmoud WM, Davis EM, Coughlin RW (1996) Biodegradation of polyurethane coatings by hydrocarbon-degrading bacteria. Int Biodeterio Biodegrad 37:69–79CrossRefGoogle Scholar
  16. Fried JR (1995) Polymer science and technology. Prentice Hall PTR, Englewood CliffsGoogle Scholar
  17. Gerner-Smidt P (1992) Ribotyping of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex. J Clin Microbiol 30:2680–2685PubMedGoogle Scholar
  18. Gerner-Smidt P, Tjernberg I, Ursing J (1991) Reliability of phenotypic tests for identification of Acinetobacter species. J Clin Microbiol 29:277–282PubMedGoogle Scholar
  19. Han SJ, Back JH, Yoon MY, Shin PK, Cheong CS, Sung MH, Hong SP, Chung I, Han YS (2003) Expression and characterization of a novel enantioselective lipase from Acinetobacter species SY-01. Biochimie 85:501–510PubMedCrossRefGoogle Scholar
  20. Howard GT, Blake RC (1999) Growth of Pseudomonas fluorescens on a polyester-polyurethane and the purification and characterization of a polyurethanase-protease enzyme. Int Biodeterio Biodegrad 42:213–220CrossRefGoogle Scholar
  21. Howard GT, Ruiz C, Hilliard N (1998) Growth of Pseudomonas chlororaphis on a polyester-polyurethane and the purification and characterization of a polyurethanase-esterase enzyme. Int Biodeterio Biodegrad 43:7–12CrossRefGoogle Scholar
  22. Howard GT, Crother B, Vicknair J (2001) Cloning, nucleotide sequencing and characterization of a polyurethanase gene (pueB) from Pseudomonas chlororaphis. Int Biodeterio Biodegrad 47:141–149CrossRefGoogle Scholar
  23. Howard GT, Duos B, Watson E (2010) Characterization of the soil microbial community associated with the decomposition of a swine carcass. Int Biodeterio Biodegrad 64:300–304CrossRefGoogle Scholar
  24. Ibrahim A, Gerner-Smidt P, Liesack W (1997) Phylogenetic relationship of the twenty-one DNA groups the genus Acinetobacter as revealed by 16 s ribosomal DNA sequence analysis. J Bacterial 47:837–841CrossRefGoogle Scholar
  25. Ishii S, Koki J, Unno H, Hori K (2004) Two morphological types of cell appendages on a strongly adhesive bacterium, Acinetobacter sp. Strain Tol 5. Appl Environ Microbiol 70:5026–5029PubMedCrossRefGoogle Scholar
  26. Jaeger KE, Ransac S, Dijkstra BW, Colson C, Van Heuvel M, Misset O (1994) Bacterial lipases. FEMS Microbiol Rev 15:29–63PubMedCrossRefGoogle Scholar
  27. Kawai F (1995) Breakdown of plastics and polymers by microorganisms. Adv Biochem Eng Biotechnol 52:151–194PubMedGoogle Scholar
  28. Knight GC, Seviouri RJ, Soddell OJA, McDonnell S, Bayl RC (1995) Metabolic variation among strains of Acinetobacter isolated from activated sludge. Water Res 29:2081–2084CrossRefGoogle Scholar
  29. Kok RG, Christoffels VM, Vosman B, Hellingwerf KJ (1993) Growth-phase dependent expression of the lipolytic system of Acinetobacter calcoaceticus BD413: cloning of a gene encoding one of the esterases. J Gen Microbiol 139:2329–2342PubMedCrossRefGoogle Scholar
  30. Kok RG, van Thor JJ, Nugteren-Roodzant IM, Brouwer MBW, Egmond MR, Nudel CB, Vosman B, Hellingwerf KJ (1995a) Characterization of the extracellular lipase, LipA, of Acinetobacter calcoaceticus BD413 and sequence analysis of the cloned structural gene. Mol Microbiol 15:803–818PubMedCrossRefGoogle Scholar
  31. Kok RG, van Thor JJ, Nugteren-Roodzant IM, Vosman B, Hellingwerf KJ (1995b) Characterization of lipase-deficient mutants of Acinetobacter calcoaceticus BD413: identification of a periplasmic lipase chaperone essential for the production of extracellular lipase. J Bacteriol 177:3295–3307PubMedGoogle Scholar
  32. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680–685CrossRefGoogle Scholar
  33. Nakajima-Kambe T, Onuma F, Kimpara N, Nakahara T (1995) Isolation and characterization of a bacterium which utilizes polyester polyurethane as a sole carbon and nitrogen source. FEMS Microbiol Lett 129:39–42PubMedCrossRefGoogle Scholar
  34. Nakajima-Kambe T, Onuma F, Akutsu Y, Nakahara T (1997) Determination of the polyester polyurethane breakdown products and distribution of the polyurethane degrading enzyme of Comamonas acidovorans strain TB-35. J Ferment Bioeng 83:456–460CrossRefGoogle Scholar
  35. Peleg AY, Seifert H, Paterson DL (2008) Acinetobacter baumannii: emergence of a successful pathogen. Clin Microbiol Rev 21:538–582PubMedCrossRefGoogle Scholar
  36. Persson B, Bentsson-Olivecrona G, Enerback S, Olivecrona T, Jornvall H (1989) Structure features of lipoprotein lipase: lipase family relationships, binding interactions, non-equivalence of lipase cofactors, vitellogenin similarities and functional subdivision of lipoprotein lipase. Eur J Biochem 179:39–45PubMedCrossRefGoogle Scholar
  37. Rowe L, Howard GT (2002) Growth of Bacillus subtilis on polyurethane and the purification and characterization of a polyurethanase-lipase enzyme. Int Biodeterio Biodegrad 50:33–40CrossRefGoogle Scholar
  38. Saunders JH, Frisch KC (1964) Polyurethanes: chemistry and technology, part II: technology. Interscience Publishers, New YorkGoogle Scholar
  39. Seifert H, Strate A, Schulze A, Pulverer G (1993) Vascular catheterrelated bloodstream infection due to Acinetobacter johnsonii (formerly Acinetobacter calcoaceticus var. lwoffii): report of 13 cases. Clin Infect Dis 17:632–636PubMedCrossRefGoogle Scholar
  40. Shannon MJR, Unterman R (1993) Evaluating bioremediation: distinguishing fact from fiction. Ann Rev Microbiol 47:715–738CrossRefGoogle Scholar
  41. Stern RS, Howard GT (2000) The polyester polyurethanase gene (pueA) from Pseudomonas chlororaphis encodes a lipase. FEMS Microbiol Lett 185:163–168PubMedCrossRefGoogle Scholar
  42. Sullivan ER, Leahy JG, Colwell RR (1999) Cloning and sequence analysis of the lipase and lipase chaperone encoding genes from Acinetobacter calcoaceticus RAG-1, and redefinition of a proteobacterial lipase family and an analogous lipase chaperone family. Gene 230:277–285PubMedCrossRefGoogle Scholar
  43. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599PubMedCrossRefGoogle Scholar
  44. Towner KJ (1997) Clinical importance and antibiotic resistance of Acinetobacter spp. J Med Microbiol 46:721–746PubMedCrossRefGoogle Scholar
  45. Uhlig K (1999) Discovering polyurethanes. Hanser Publisher, MunichGoogle Scholar
  46. Urbanski J, Czerwinski W, Janicka K, Majewska F, Zowall H (1977) Handbook of analysis of synthetic polymers and plastics. Ellis Horwood Limited, ChichesterGoogle Scholar
  47. Vega R, Main T, Howard GT (1999) Cloning and expression in Escherichia coli of a polyurethane-degrading enzyme from Pseudomonas fluorescens. Int Biodeterio Biodegrad 43:49–55CrossRefGoogle Scholar
  48. Winkler FK, D’Arcy A, Hunzinger W (1990) Structure of human pancreatic lipase. Nature (London) 343:7–13CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Gary T. Howard
    • 1
  • William N. Norton
    • 1
  • Timothy Burks
    • 1
  1. 1.Department of Biological SciencesSoutheastern Louisiana UniversityHammondUSA

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