Applied Microbiology and Biotechnology

, Volume 77, Issue 5, pp 1139–1144 | Cite as

Azoreductase and dye detoxification activities of Bacillus velezensis strain AB

  • Amit Bafana
  • Tapan Chakrabarti
  • Sivanesan Saravana Devi
Environmental Biotechnology


Azo dyes are known to be a very important and widely used class of toxic and carcinogenic compounds. Although lot of research has been carried out for their removal from industrial effluents, very little attention is given to changes in their toxicity and mutagenicity during the treatment processes. Present investigation describes isolation of a Bacillus velezensis culture capable of degrading azo dye Direct Red 28 (DR28). Azoreductase enzyme was isolated from it, and its molecular weight was found to be 60 kDa. The enzyme required NADH as cofactor and was oxygen-insensitive. Toxicity and mutagenicity of the dye during biodegradation was monitored by using a battery of carefully selected in vitro tests. The culture was found to degrade DR28 to benzidine and 4-aminobiphenyl, both of which are potent mutagens. However, on longer incubation, both the compounds were degraded further, resulting in reduction in toxicity and mutagenicity of the dye. Thus, the culture seems to be a suitable candidate for further study for both decolourization and detoxification of azo dyes, resulting in their safe disposal.


Direct red 28 Azoreductase Zymogram Ames test Biodegradation 

Supplementary material

253_2007_1212_Fig1_ESM.gif (26 kb)
Fig. S1

Characterization of azoreductase enzyme from B. velezensis. a Zymogram showing presence of azoreductase as clear band, b SDS-PAGE gel showing M.W. of azoreductase from the zymogram (GIF 26 kb)

253_2007_1212_Fig1_ESM.tif (31 kb)
High resolution image file (TIF 32 kb)
253_2007_1212_Fig2_ESM.gif (33 kb)
Fig. S2

a HPLC chromatogram and b mass spectrum of standard 4-ABP, c HPLC chromatogram and d mass spectrum of standard benzidine (GIF 37 kb)

253_2007_1212_Fig2_ESM.tif (30 kb)
High resolution image file (TIF 30 kb)


  1. Alexander E, Pham D, Steck TR (1999) The viable but nonculturable condition is induced by copper in Agrobacterium tumefaciens and Rhizobium leguminosarum. Appl Environ Microbiol 65:3754–3756Google Scholar
  2. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (1987) Current protocols in molecular biology. New York, WileyGoogle Scholar
  3. Bafana A, Devi SS, Krishnamurthi K, Chakrabarti T (2007) Kinetics of decolourisation and biotransformation of direct black 38 by C. hominis and P stutzeri. Appl Microbiol Biotechnol 74:1145–1152CrossRefGoogle Scholar
  4. Botsford JL (1998) A simple assay for toxic chemicals using a bacterial indicator. World J Microbiol Biotechnol 14:369–376CrossRefGoogle Scholar
  5. Chen H (2006) Recent Advances in Azo Dye Degrading Enzyme Research. Curr Protein Pept Sci 7:101–111CrossRefGoogle Scholar
  6. Chung KT, Cerniglia CE (1992) Mutagenicity of azo dyes: structure-activity relationships. Mutat Res 277:201–220Google Scholar
  7. Edwards U, Rogall T, Blöcker H, Emde M, Böttger EC (1989) Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA. Nucleic Acids Res 17:7843–7853CrossRefGoogle Scholar
  8. Freshney RI (1983) Culture of Animal Cells. A manual of basic techniques. New York, Alan Liss IncGoogle Scholar
  9. Hu TL (2001) Kinetics of azoreductase and assessment of toxicity of metabolic products from azo dyes by Pseudomonas luteola. Water Sci Technol 43:261–269Google Scholar
  10. Isik M, Sponza DT (2004) Monitoring of toxicity and intermediates of C.I. Direct Black 38 azo dye through decolorization in an anaerobic/aerobic sequential reactor system. J Hazard Mater 114:29–39CrossRefGoogle Scholar
  11. Işik M, Sponza DT (2006) Fate and toxicity of azo dye metabolites under batch long-term anaerobic incubations. Enzyme Microb Technol 40:934–939Google Scholar
  12. Kulla HG, Klausener F, Meyer U, Lüdeke B, Leisinger T (1983) Interference of aromatic sulfo groups in the microbial degradation of the azo dyes Orange I and Orange II. Arch Microbiol 135:1–7CrossRefGoogle Scholar
  13. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefGoogle Scholar
  14. Maier J, Kandelbauer A, Erlacher A, Cavaco-Paulo A, Gubitz M (2004) A new alkali-thermostable azoreductase from Bacillus sp. strain SF. Appl Environ Microbiol 70:837–844CrossRefGoogle Scholar
  15. Maron D, Ames B (1983) Revised Methods for the Salmonella mutagenicity test. Mutat Res 113:173–215Google Scholar
  16. Morgan DL, Dunnick JK, Goehl T, Jokinen MP, Matthews HB, Zeiger E, Mennear JH (1994) Summary of the National Toxicology Program Benzidine Dye Initiative. Environ Health Perspect 102:63–78CrossRefGoogle Scholar
  17. Novotný Č, Dias N, Kapanen A, Malachová K, Vándrovcová M, Itävaara M, Lima N (2006) Comparative use of bacterial, algal and protozoan tests to study toxicity of azo- and anthraquinone dyes. Chemosphere 63:1436–1442CrossRefGoogle Scholar
  18. Pourbabaee AA, Malekzadeh F, Sarbolouki MN, Najafi F (2006) Aerobic decolorization and detoxification of a disperse dye in textile effluent by a new isolate of Bacillus sp. Biotechnol Bioeng 93:631–635CrossRefGoogle Scholar
  19. van der Zee FP, Villaverde S (2005) Combined anaerobic–aerobic treatment of azo dyes—A short review of bioreactor studies. Water Res 39:1425–1440CrossRefGoogle Scholar
  20. Zimmermann T, Kulla HG, Leisinger T (1982) Properties of purified orange II azoreductase, the enzyme initiating azo dye degradation by Pseudomonas KF46. Eur J Biochem 129:197–203CrossRefGoogle Scholar
  21. Zorrilla I, Balebona MC, Moriñigo MA (2001) Adaptation of an [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay to evaluate the cytotoxicity of the extracellular products of micro-organisms pathogenic to fish. Lett Appl Microbiol 33:329–333CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Amit Bafana
    • 1
  • Tapan Chakrabarti
    • 1
  • Sivanesan Saravana Devi
    • 1
  1. 1.Environmental Biotechnology DivisionNational Environmental Engineering Research Institute (NEERI)NagpurIndia

Personalised recommendations