, Volume 25, Issue 7, pp 4179–4192 | Cite as

Biobleaching of waste paper using lignolytic enzyme from Fusarium equiseti VKF2: a mangrove isolate

  • Vinod Kumar Nathan
  • Subha Rajam Kanthimathinathan
  • Mary Esther Rani
  • Gunaseeli Rathinasamy
  • N. D. Kannan
Original Paper


Laccase is a major enzyme used in the paper and pulp industries for bio-bleaching applications. This paper focused on the bio-bleaching property of a potential lignolytic fungus, Fusarium equiseti VKF-2. Optimum conditions for laccase production were tested by statistical models under solid state fermentation using saw dust as substrate. Fusarium equiseti VKF-2 achieved a maximum laccase activity of 305 Ug−1 of substrate. During purification, 67% yield was obtained with 80% ammonium sulphate. The enzyme activity was confirmed through zymogram analysis with 2 active fractions observed at 94 and 75 kDa. Laccase treatment resulted in defibrillations, crack formation and changes in the functional groups of paper pulp. The laccase facilitated the maximum hexenuronic acid release with Kappa number reduction. There was a Δ brightness of 15% following 4 h of enzymatic treatment. Hence, the present laccase was found to be suitable for the bio-bleaching of newspaper waste via an eco-friendly process.

Graphical abstract


Laccase Fusarium equiseti Deinking Brightness Optimization 



Authors are thankful to Department of Science and Technology, Government of India and TNSCST for providing the grant to facilitate the research [Sanction Order No. DST/SSTP/TN/2K 10/126(G) 13-09-2011]. NVK thank Science and Engineering Research Board, Government of India for National Post Doctorate Fellowship [PDF/2016/00438].

Supplementary material

10570_2018_1834_MOESM1_ESM.docx (909 kb)
Supplementary material 1 (DOCX 909 kb)


  1. Bajpai P, Bajpai PK (1998) Deinking with enzymes: a review. Tappi J 81:111–117Google Scholar
  2. Box GE, Behnken DW (1960) Some new three level designs for the study of quantitative variables. Technometrics 2:455–475CrossRefGoogle Scholar
  3. Bucher VVC, Hyde KD, Pointing SB, Reddy CA (2004) Production of wood decay enzymes, mass loss and lignin solubilization in wood by marine ascomycetes and their anamorphs. Fungal Diversity 15:1–14Google Scholar
  4. Carvalho MG, Ferreira PJ, Figueiredo MM (2000) Cellulose depolymerisation and paper properties in E. globulus kraft pulps. Cellulose 7:359–368CrossRefGoogle Scholar
  5. Casimir-Schenkel J, Davis S, Fiechter A, Gysin B, Murray E, Perrolaz JJ, Zimmermann W, Sandoz Ltd (1995) Pulp bleaching with thermo-stable xylanase of Thermomonospora fusca. US Patent 5: 407,827Google Scholar
  6. Chai XS, Zhu J, Luo Q, Yoon SH (2001) The fate of hexenuronic acid groups during kraft pulping of hardwoods. Tappi J 27:1–4Google Scholar
  7. Chawachart N, Khanongnuch C, Watanabe T, Lumyong S (2004) Rice bran as an efficient substrate for laccase production from thermotolerant basidiomycete Coriolus versicolor strain RC3. Fungal Divers 15:23–32Google Scholar
  8. Chhaya U, Gupte A (2010) Optimization of media components for laccase production by litter dwelling fungal isolate Fusarium incarnatum LD-3. J Basic Microbiol 50:43–51CrossRefPubMedGoogle Scholar
  9. Chutani P, Sharma KK (2015) Biochemical evaluation of xylanases from various filamentous fungi and their application for the deinking of ozone treated newspaper pulp. Carbohydr Polym 127:54–63CrossRefPubMedGoogle Scholar
  10. Chutani P, Sharma KK (2016) Concomitant production of xylanases and cellulases from Trichoderma longibrachiatum MDU-6 selected for the deinking of paper waste. Bioprocess Biosyst Eng 39:747–751CrossRefPubMedGoogle Scholar
  11. D’Souza-Ticlo D, Sharma D, Raghukumar C (2009) A thermostable metal-tolerant laccase with bioremediation potential from a marine-derived fungus. Mar Biotechnol 11:725–737CrossRefPubMedGoogle Scholar
  12. Desai SS, Nityanand C (2011) Microbial laccases and their applications: a review. Asian J Biotechnol 3:98–124CrossRefGoogle Scholar
  13. Ding Z, Chen Y, Xu Z, Peng L, Xu G, Gu Z, Zhang L, Shi G, Zhang K (2014) Production and characterization of laccase from Pleurotus ferulae in submerged fermentation. Ann Microbiol 1:121–129CrossRefGoogle Scholar
  14. Fernaud JH, Carnicero A, Perestelo F, Cutuli MH, Arias E, Falcón MA (2006) Upgrading of an industrial lignin by using laccase produced by Fusarium proliferatum and different laccase–mediator systems. Enzym Microb Technol 38:40–48CrossRefGoogle Scholar
  15. Gonzalez JC, Medina SC, Rodriguez A, Osma JF, Alméciga-Díaz CJ, Sánchez OF (2013) Production of Trametes pubescens laccase under submerged and semi-solid culture conditions on agro-industrial wastes. PLoS ONE 8:e73721CrossRefPubMedPubMedCentralGoogle Scholar
  16. Hankin L, Anagnostakis SL (1975) The use of solid media for detection of enzyme production by fungi. Mycologia 67:597–607CrossRefGoogle Scholar
  17. Jayant M, Rashmi J, Shailendra M, Deepesh Y (2011) Production of cellulase by different co-culture of Aspergillus niger and Penicillium chrysogenum from waste paper, cotton waste and baggase. J Yeast Fungal Res 2:24–27Google Scholar
  18. Jeffries TW, Klungness JH, Sykes MS, Rutledge-Cropsey KR (1994) Comparison of enzyme-enhanced with conventional deinking of xerographic and laser-printed paper. Tappi J 77:173–179Google Scholar
  19. Kurtzman CP, Robnett CJ (1997) Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5′ end of the large-subunit (26S) ribosomal DNA gene. J Clin Microbiol 35:1216–1223PubMedPubMedCentralGoogle Scholar
  20. Kwiatos N, Ryngajłło M, Bielecki S (2015) Diversity of laccase-coding genes in Fusarium oxysporum genomes. Front Microbiol 6:933CrossRefPubMedPubMedCentralGoogle Scholar
  21. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefPubMedGoogle Scholar
  22. Maity C, Ghosh K, Halder SK, Jana A, Adak A, Das Mohapatra PK, Pati BR, Mondal KC (2012) Xylanase isozymes from the newly isolated Bacillus sp. CKBx1D and optimization of its deinking potentiality. Appl Biochem Biotechnol 167:1208–1219CrossRefPubMedGoogle Scholar
  23. Melo SC, Pungartnik C, Cascardo JC, Brendel M (2006) Rapid and efficient protocol for DNA extraction and molecular identification of the basidiomycete Crinipellis perniciosa. Genet Mol Res 5:851–855PubMedGoogle Scholar
  24. Moubasher MH, Abdel-Hafez SII, Abdel-Fattah HM, Mohanram AM (1982) Direct estimation of cellulose, hemicellulose and lignin. J Agric Res 46:1467–1476Google Scholar
  25. Nathan VK, Rani ME, Rathinasamy G, Dhiraviam KN, Jayavel S (2014a) Process optimization and production kinetics for cellulase production by Trichoderma viride VKF3. SpringerPlus 3:92CrossRefPubMedPubMedCentralGoogle Scholar
  26. Nathan VK, Rani ME, Rathinasamy G, Kannan ND (2014b) Potential of xylanase from Trichoderma viride VKF3 in waste paper pulp characteristics modification. In: Proceeding of international conference on chemical, environment and biological sciences, Kuala Lumpur, Malaysia, pp 54–60.
  27. Nathan VK, Rani ME, Rathinasamy G, Dhiraviam KN (2017) Low molecular weight xylanase from Trichoderma viride VKF3 for bio-bleaching of newspaper pulp. BioResources 12:5264–5278CrossRefGoogle Scholar
  28. Nathan VK, Rani ME, Rathinasamy G, Dhiraviam KN (2018) Paper pulp modification and deinking efficiency of cellulase-xylanase complex from Escherichia coli SD5. Int J Biol Macromol 111:289–295CrossRefGoogle Scholar
  29. Octavio LC, Irma PPMC, Ricardo BRJ, Francisco VO (2006) Laccase. In: Guevara-Gonzalez RG, Torres-Pacheco I (eds) Advances in agricultural and food biotechnology. Departmento de Ingenieria Bioquimica, Instituto Technologico de Celaya, Kerala, pp 323–340Google Scholar
  30. Pala H, Mota M, Gama FM (2006) Factors influencing MOW deinking: laboratory scale studies. Enzym Microb Technol 38:81–87CrossRefGoogle Scholar
  31. Pathak P, Bhardwaj NK, Singh AK (2014) Production of crude cellulase and xylanase from Trichoderma harzianum PPDDN10 NFCCI-2925 and its application in photocopier waste paper recycling. Appl Biochem Biotechnol 172:3776–3797CrossRefPubMedGoogle Scholar
  32. Patrick F, Mtui G, Mshandete AM, Johansson G, Kivaisi A (2009) Purification and characterization of laccase from the basidiomycete Funalia trogii (Berk.) isolated in Tanzania. Afr J Biochem Res 3:250–258Google Scholar
  33. Plackett RL, Burman JP (1946) The design of optimum multifactorial experiments. Biometrika 33:305–325CrossRefGoogle Scholar
  34. Raghukumar C, Muraleedharan U, Gaud VR, Mishra R (2004) Xylanases of marine fungi of potential use for biobleaching of paper pulp. J Ind Microbial Biotechnol 31:433–441CrossRefGoogle Scholar
  35. Rajan A, Kurup JG, Abraham TE (2010) Solid state production of manganese peroxidases using arecanut husk as substrate. Braz Arch Biol Technol 53:555–562CrossRefGoogle Scholar
  36. Sealey J, Ragauskas AJ (1998) Investigation of laccase/N-hydroxybenzotriazole delignification of kraft pulp. J Wood Chem Technol 18:403–416CrossRefGoogle Scholar
  37. Srinivasan MC, Rele MV (1999) Microbial xylanases for paper industry. Curr Sci 77:137–142Google Scholar
  38. Stoilova I, Krastanov A, Stanchev V (2010) Properties of crude laccase from Trametes versicolor produced by solid-substrate fermentation. Adv Biosci Biotechnol 1:208–215CrossRefGoogle Scholar
  39. TAPPI T236 om-99 (2004) Kappa number of pulp. TAPPI press, AtlantaGoogle Scholar
  40. Valls C, Colom JF, Baffert C, Gimbert I, Roncero MB, Sigoillot JC (2010) Comparing the efficiency of the laccase–NHA and laccase–HBT systems in eucalyptus pulp bleaching. Biochem Eng J 49:401–407CrossRefGoogle Scholar
  41. Vivekanand V, Dwivedi P, Pareek N, Singh RP (2011) Banana peel: a potential substrate for laccase production by Aspergillus fumigatus VkJ2. 4.5 in solid-state fermentation. Appl Biochem Biotechnol 165:204CrossRefPubMedGoogle Scholar
  42. Vuorinen T, Buchert J, Teleman A, Tenkanen M, Fagerstrom P (1996) Selective hydrolysis of hexenuronic acid groups and its application in ECF and TCF bleaching of kraft pulps. In: Proceedings of the international pulp bleaching conference, vol 1. Atlanta, GA, 14 April, 1996Google Scholar
  43. Woldesenbet F, Gupta N, Sharma P (2012) Statistical optimization of the production of a cellulase-free, thermo-alkali-stable, salt-and solvent-tolerant xylanase from Bacillus halodurans by solid state fermentation. Arch Appl Sci Res 4:524–535Google Scholar
  44. Woldesenbet F, Virk AP, Gupta N, Sharma P (2013) Biobleaching of mixed wood kraft pulp with alkalophilic bacterial xylanase, mannanase and laccase-mediator system. J Microbiol Biotechnol Res 3:32–41Google Scholar
  45. Zhang X, Renaud S, Paice M (2008) Cellulase deinking of fresh and aged recycled newsprint/magazines (ONP/OMG). Enzym Microb Technol 43:103–108CrossRefGoogle Scholar
  46. Zouari-Mechichi H, Mechichi T, Dhouib A, Sayadi S, Martínez AT, Martínez MJ (2006) Laccase purification and characterization from Trametes trogii isolated in Tunisia: decolorization of textile dyes by the purified enzyme. Enzym Microb Technol 39:141–148CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  • Vinod Kumar Nathan
    • 1
    • 2
  • Subha Rajam Kanthimathinathan
    • 2
  • Mary Esther Rani
    • 2
  • Gunaseeli Rathinasamy
    • 2
  • N. D. Kannan
    • 3
  1. 1.Regional CentreNational Institute of Oceanography-CSIRCochinIndia
  2. 2.Department of Botany and MicrobiologyLady Doak CollegeMaduraiIndia
  3. 3.Department Plant Biotechnology, School of BiotechnologyMadurai Kamaraj UniversityMaduraiIndia

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