Indian Journal of Microbiology

, Volume 52, Issue 4, pp 701–707 | Cite as

The Determination of Assay for Laccase of Bacillus subtilis WPI with Two Classes of Chemical Compounds as Substrates

  • Fatemeh SheikhiEmail author
  • Mohammad Roayaei Ardakani
  • Naeimeh Enayatizamir
  • Susana Rodriguez-Couto
Original Article


Ligninolytic enzyme complexes are involved in lignin degradation. Among them laccases are outstanding because they use molecular oxygen as a co-substrate instead of hydrogen peroxide as used by peroxidases. Bacterial laccase of Bacillus genus was first reported in Claus and Filip (Microbiol Res 152:209–216, 1997), since then more bacterial laccases have been found. In this research, laccase-producing bacteria were screened from pulp and paper industry wastewater, bagass and sugarcane rhizosphere. Nutrient agar medium containing 0.5 mM of guaiacol was used. It was observed that the laccase-producing strains developed brown colour from which 16 strains of Bacillus were identified. One of the isolated strains was identified as Bacillus subtilis WPI based on the results of biochemical tests and 16S rDNA sequence analysis. This strain showed laccase-like activity towards the oxidizing substrates ABTS and guaiacol. In this study guaiacol was used as the substrate of laccase activity assay. For determination of laccase activity of this isolate guaiacol was used as a substrate of assay for the first time in this study. SDS-PAGE and Native-PAGE confirmed the presence of laccase.


Laccase Bacillus subtilis Pulp and paper wastewater Isolation Guaiacol 



This research was supported by Biotechnology and Biological Research Center of Shahid Chamran University of Ahvaz (Iran).


  1. 1.
    Claus H, Filip Z (1997) The evidence of a laccase-like enzyme activity in a Bacillus sphaericus strain. Microbiol Res 152:209–216CrossRefGoogle Scholar
  2. 2.
    Thurston CF (1994) The structure and function of fungal laccases. Microbiology 140:19–26CrossRefGoogle Scholar
  3. 3.
    Desai SS, Nityanand C (2011) Microbial laccases and their application. Asian J Biotechnol 3:98–124CrossRefGoogle Scholar
  4. 4.
    Pazarlıoglu NK, Sariisik M, Telefoncu A (2005) Laccase: production by Trametes versicolor and application to denim washing. Process Biochem 40(1):673–1678Google Scholar
  5. 5.
    Xu F (2005) Applications of oxidoreductases: recent progress. Ind Biotechol 1:38–50CrossRefGoogle Scholar
  6. 6.
    Gardiol AE, Hernandez RJ, Harte BR (1998) Device for detecting oxygen with oxidase, US Pat. 5, 804, 401Google Scholar
  7. 7.
    Faure D, Bouillant ML, Bally R (1994) Isolation of Azospirillum lipoferum 4T mutants affected in melanization and laccase activity. Appl Environ Microbiol 60:3413–3415PubMedGoogle Scholar
  8. 8.
    Endo K, Hosono K, Beppu T, Ueda K (2002) A novel extra cytoplasmatic phenol oxidase of Streptomyces: its possible involvement in the onset of morphogenesis. Microbiology 148:1767–1776PubMedGoogle Scholar
  9. 9.
    Hullo MF, Moszer I, Danchin A, Martin-Verstraete I (2001) CotA of Bacillus subtilisis a copper-dependent laccase. J Bacteriol 183:5426–5430PubMedCrossRefGoogle Scholar
  10. 10.
    Augustine AJ, Kragh ME, Sarangi R, Fujii S, Liboiron BD, Stoj CS, Kosman DJ, Hodgson KO, Hedman B, Solomon EI (2008) Spectroscopic studies of perturbed T1 Cu sites in the multicopper oxidases Saccharmycescerevisiae Fet3p and Rhusvernicifera laccase: allosteric coupling between the T1 and trinuclear Cu sites. Biochemistry 47:2036–2045PubMedCrossRefGoogle Scholar
  11. 11.
    Rodgers CJ, Blanford CF, Giddens SR, Skamnioti P, Armstrong FA, Gurr SJ (2010) Designer laccases: a vogue for high-potential fungal enzymes? Trends Biotechnol 28:63–72PubMedCrossRefGoogle Scholar
  12. 12.
    Koschorreck K, Schmid RD, Urlacher VB (2009) Improving the functional expression of a Bacillus licheniformis laccase by random and site-directed mutagenesis. BMC Biotechnol 9:12–21PubMedCrossRefGoogle Scholar
  13. 13.
    BadoeiDalfard A, Khajeh K, Soudi MR, Naderi-Manesh H, Ranjbar B, Hassan Sajed R (2006) Isolation and biochemical characterization of laccase and tyrosinase activities in a novel melanogenic soil bacterium. Enzyme Microb Technol 39:1409–1416CrossRefGoogle Scholar
  14. 14.
    Bains J, Capalash N, Sharma P (2003) Laccase from a non-melanogenic, alkalotolerantγ-proteobacterium JB isolated from industrial wastewater drained soil. Biotechnol Lett 25(1):155–1159CrossRefGoogle Scholar
  15. 15.
    Machado KMG, Matheus DR (2006) “Potential of a ligninolytic enzymatic complex produced by Pleurotusostreatus during growth on solid substrate for the biodegradation of organic pollutants”Brazilian. J Microbiol 37:468–473Google Scholar
  16. 16.
    Niku-Paavola ML, Raaska L, Itvaara M (1990) Detection of white-rot fungi by a nontoxic stain. Mycol Res 94:27–31CrossRefGoogle Scholar
  17. 17.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacterio phage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  18. 18.
    Papizadeh M, Roayaei Ardakani M, Ebrahimipour Gh, Motamedi H (2010) Utilization of dibenzothiophene as sulfur source by Microbacterium sp. NISOC-06. World J Microbiol Biotechnol 26:1195–1200CrossRefGoogle Scholar
  19. 19.
    Duarte GF, Rosado AS, Seldin L, De Araujo W, Van Elsas JD (2001) Analysis of bacterial community structure in sulfurous-oil containing soils and detection of species carrying dibenzothiophene desulfurization (dsz) genes. Appl Environ Microbiol 67:1052–1062PubMedCrossRefGoogle Scholar
  20. 20.
    Liers C, Ullrich R, Pecyna M, Schlosser D, Hofrichter M (2007) Production, purification and partial enzymatic and molecular characterization of a laccase from the wood-rotting ascomycete Xylariapolymorpha. Enzyme Microbial Technol 41:785–793CrossRefGoogle Scholar
  21. 21.
    Li A, Zhu Y, Xu L, Zhu W, Tian X (2008) Comparative study on the determination of assay for laccase of Trametes sp. Af J Biochem Res 2:181–183Google Scholar
  22. 22.
    Robles A, Lucas R, Cienfuegos GA, Galvez A (2000) Phenol-oxidase (laccase) activity in strains of the hyphomycete Chalaraparadoxa isolated from olive mill wastewater disposal ponds. Enzyme Microb Technol 26:484–490PubMedCrossRefGoogle Scholar
  23. 23.
    Ruijssenaars HJ, Hartmans S (2004) A cloned Bacillus halodurans multicopper oxidase exhibiting alkaline laccase activity. Appl Microbiol Biotechnol 65:177–182PubMedCrossRefGoogle Scholar
  24. 24.
    Niladevi KN, Jacob N, Prema P (2008) Evidence for a halotolerant-alkaline laccase in Streptomyces psammoticus Purification and characterization. Process Biochem 43:654–660CrossRefGoogle Scholar
  25. 25.
    Mohammadian M, Fathi-Roudsari M, Mollania N, Badoei-Dalfard A, Khajeh K (2010) Enhanced expression of a recombinant bacterial laccase at low temperature and microaerobic conditions: purification and biochemical characterization. J Ind Microbiol Biotechnol 37:863–869PubMedCrossRefGoogle Scholar
  26. 26.
    Reiss R, Ihssen J, Thony-Meyer L (2011) Bacillus pumilus laccase: a heat stable enzyme with a wide substrate spectrum. BMC Biotechnol 11:1–11CrossRefGoogle Scholar

Copyright information

© Association of Microbiologists of India 2012

Authors and Affiliations

  • Fatemeh Sheikhi
    • 1
    Email author
  • Mohammad Roayaei Ardakani
    • 1
  • Naeimeh Enayatizamir
    • 2
  • Susana Rodriguez-Couto
    • 3
    • 4
  1. 1.Department of Biology, Faculty of ScienceUniversity of Shahid ChamranAhvazIran
  2. 2.Department of Soil Science, Faculty of AgricultureUniversity of Shahid ChamranAhvazIran
  3. 3.CEIT, Section of Environmental EngineeringSan SebastiánSpain
  4. 4.IKERBASQUE, Basque Foundation for ScienceBilbaoSpain

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