A novel thermo-alkali stable catalase–peroxidase from Oceanobacillus oncorhynchi subsp. incaldaniensis: purification and characterization

  • Valeria Calandrelli
  • Agata Gambacorta
  • Ida Romano
  • Vito Carratore
  • Licia Lama
Original Paper

Abstract

A novel thermo-alkali-stable catalase–peroxidase from Oceanobacillus oncorhynchi subsp. incaldaniensis subsp. nov., strain 20AG, was purified and characterized. The protein purified from the cells resulted in 110-fold purification with a specific activity of 35,000 U/mg. The enzyme consisted of four identical subunits of 72 kDa as determined by SDS-PAGE and the total molecular mass measured by gel filtration was 280 kDa. The heme content was determined to be 1 heme per homodimer. The enzyme showed a Soret peak at 406 nm in the oxidized form and was easily reduced by dithionite. The enzyme showed an appreciable peroxidase activity in addition to high catalase activity. The behaviour of this heme-enzyme was typical of the class of prokaryotic catalase–peroxidases, which are sensitive to cyanide and insensitive to the eukaryotic catalase inhibitor 3-amino-1,2,4-triazole. The enzyme was active over a temperature range from 30 to 60°C and a pH range from 5 to 10, with an optimum pH about 9.0 and an optimum temperature of 40°C. The enzyme was stable in the pH range of 5.0 to 10.0 after 1 h of treatment at 40°C. The enzyme was stable for 24 h at 40°C with a half-life of 4 h 60°C. The enzyme had a Km of 24 mM for hydrogen peroxide. The amino terminal amino acid sequence of the catalase–peroxidase from strain 20AG was SEKRKMTTAFGA and it showed no homology with other catalases.

Keywords

Alkalitolerant Bacterium Catalase Catalase–peroxidase Halophilic 

References

  1. Beers RF Jr, Sizer IW (1952) A spetrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J Biol Chem 195:133–140Google Scholar
  2. Brown-Paterson NJ, Salin ML (1995) Purification and characterization of a mesohalic catalase from the halophilic bacterium Halobacterium halobium. J Bacteriol 177:378–384 Google Scholar
  3. Claiborne A, Fridovich I (1979) Purification of the o-dianisidine peroxidase from Escherichia coli B. J Biol Chem 254(10):4245–4252Google Scholar
  4. Davis BJ (1964) Disc gel electrophoresis. Ann NY Acad Sci 121:404–427. doi:10.1111/j.1749-6632.1964.tb14213.x CrossRefGoogle Scholar
  5. Hildebrandt AG, Roots I (1975) Reduced nicotinamideadenine dinucleotide phosphate (NADP)-dependent formation and breakdown of hydrogen peroxide during mixed function oxidation reactions in liver microsomes. Arch Biochem Biophys 171:385–397. doi:10.1016/0003-9861(75)90047-8 CrossRefGoogle Scholar
  6. Hochman A, Goldberg I (1991) Purification and characterization of a catalase–peroxidase and a typical catalase from the bacterium Klebsiella pneumoniae. Biochim Biophys Acta 1077:299–307 Google Scholar
  7. Johnsson K, Froland WA, Schultz PG (1997) Overexpression, purification, and characterisation of the catalase-peroxidase KatG from Mycobacterium tuberculosis. J Biol Chem 272(5):2834–2840. doi:10.1074/jbc.272.5.2834 CrossRefGoogle Scholar
  8. Kim H, Lee JS, Hah YC, Roe JH (1994) Characterization of the major catalase from Streptomyces coelicolor ATCC 10147. Microbiology 140:3391–3397CrossRefGoogle Scholar
  9. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. doi:10.1038/227680a0 CrossRefGoogle Scholar
  10. Loprasert S, Negoro S, Okada H (1988) Thermostable peroxidase from Bacillus stearothermophilus. J Gen Microbiol 134:1971–1976 Google Scholar
  11. Marcinkeviciene JA, Magliozzo RS, Blanchard JS (1995) Purification and characterization of the Mycobacterium smegmatis catalase peroxidase involved in isoniazid activation. J Biol Chem 270(38):22290–22295. doi:10.1074/jbc.270.38.22290 CrossRefGoogle Scholar
  12. Matsudaira P (1987) Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem 262:10035–10038 Google Scholar
  13. Ogawa J, Sulistyaningdyan WT, Li QS, Tanaka H, Xie S-X, Kano K, Ikeda T, Shimizu S, (2004) Two extracellular proteins with alkaline peroxidase activity, a novel cytochrome c and a catalase-peroxidase, from Bacillus sp. No.13. Biochim Biophys Acta 1699:65–75 Google Scholar
  14. Phucharoen K, Hoschino K, Yakenaka Y, Shinozawa T (2002) Purification, characterization, and gene sequencing of a catalase from an alkali- and halo-tolerant bacterium, Halomonas sp. SK1. Biosci Biotechnol Biochem 66:955–962. doi:10.1271/bbb.66.955 CrossRefGoogle Scholar
  15. Regelsberger G, Obinger C, Zoder R, Altmann F, Peschek GA (1999) Purification and characterization of a hydroperoxidase from the cyanobacterium Synechocystis PCC 6803: identification of its gene by peptide mass mapping using matrix assisted laser desorption ionization time-of-flight mass spectrometry. FEMS Microbiol Lett 170:1–12. doi:10.1111/j.1574-6968.1999.tb13348.x CrossRefGoogle Scholar
  16. Rocha ER, Smith CJ (1995) Biochemical and genetic analyses of a catalase from the anaerobic bacterium Bacteroides fragilis. J Bacteriol 177:3111–3119 Google Scholar
  17. Romano I, Lama L, Nicolaus B, Poli A, Gambacorta A, Giordano A (2006) Oceanobacillus oncorhynchi subsp. incaldanensis susp. nov., an alkalitolerant halophile isolated from an algal mat collected from a sulfurous spring in Campania (Italy), and emended description of Oceanobacillus oncorhynchi. Int J Syst Evol Microbiol 56:805–810. doi:10.1099/ijs.0.63575-0 CrossRefGoogle Scholar
  18. Thompson VS, Schaller KD, Apel WA (2003) Purification and characterization of a novel thermo-alkali stable catalase from Thermus brockianus. Biotechnol Prog 19:1292–1299. doi:10.1021/bp034040t CrossRefGoogle Scholar
  19. Youn HD, Yim YI, Kim K, Hah YC, Kang SO (1995) Spectral characterization and chemical modification of catalase-peroxidase from Streptomyces sp. J Biol Chem 270(23):13740–13747. doi:10.1074/jbc.270.23.13740 CrossRefGoogle Scholar
  20. Yumoto I, Fukumori Y, Yamanaka T (1990) Purification and characterization of catalase from a facultative alkalophilic Bacillus. J Biochem 108:583–587 Google Scholar
  21. Zhang XQ, Zhao YF, Du GC, Xu ZH, Chen J, Ma YH (2005) Purification and characterization of a monofunctional catalase from an alkaliphilic Bacillus sp. F26. Sheng Wu Gong Cheng Xue Bao 21(1):71–77 Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Valeria Calandrelli
    • 1
  • Agata Gambacorta
    • 1
  • Ida Romano
    • 1
  • Vito Carratore
    • 2
  • Licia Lama
    • 1
  1. 1.Istituto di Chimica Biomolecolare CNRPozzuoliItaly
  2. 2.Istituto di Biochimica delle Proteine CNRNapoliItaly

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