BioEnergy Research

, Volume 5, Issue 2, pp 363–371 | Cite as

Isolation and Characterization of a Xylan-Degrading Enzyme from Aspergillus niger van Tieghem LPM 93 with Potential for Industrial Applications

  • Natália von Gal Milanezi
  • Diana Paola Gómez Mendoza
  • Félix Gonçalves de Siqueira
  • Luciano Paulino Silva
  • Carlos André Ornelas Ricart
  • Edivaldo Ximenes Ferreira Filho


Aspergillus niger van Tieghem LPM 93 was shown in an earlier study to produce the most thermostable β-xylanase, which was effective for improving brightness and delignification of non-delignified and oxygen-bleached samples of eucalyptus kraft pulp. Here, we report the production, purification, and characterization of a xylan-degrading enzyme (XynI) from this strain grown in submerged liquid cultivation on medium containing sugar cane bagasse as the carbon source. XynI was isolated by ultrafiltration and gel-filtration chromatography and characterized. The fungus displayed high levels of xylanolytic activity after the second day of cultivation, and this activity remained constant up to the 50th day. The molecular mass of XynI was in the range of 32–33 kDa as determined by mass spectrometry and SDS-PAGE. The two-dimensional gel electrophoresis analysis showed the existence of multiple forms of β-xylanases in XynI. XynI showed the highest activity at 50°C and pH 4.5 and was stable in sodium acetate buffer at pH 4.5. The Km and Vmax values were 47.08 mg/ml and 3.02 IU/ml, respectively. Salts inhibited the activity of XynI to different degrees. N-Bromosuccinimide caused marked inhibition of XynI. On the other hand, β-mercaptoethanol and l-tryptophan were the best enzyme activators.


Aspergillus niger Sugar Cane Bagasse β-Xylanase Isoforms 


  1. 1.
    Bissoon S, Singh S, Christov L (2002) Evaluation of the bleach-enhancing effect of xylanases on bagasse pulp. Progress Biotechnol 2:247–254Google Scholar
  2. 2.
    Blum H, Beier H, Gross B (1987) Improved silver staining of plant proteins, RNA and DNA in polyacrilamide gels. Electrophoresis 8:93–99Google Scholar
  3. 3.
    Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedGoogle Scholar
  4. 4.
    Chidi SB, Godana B, Ncube I, van Rensburg EJ, Cronshaw A, Abotsi EK (2008) Production, purification and characterization of cellulase-free xylanase from Aspergillus terreus UL 4209. African J Biotechnol 7:3939–3948Google Scholar
  5. 5.
    Collins T, Gerday C, Feller G (2005) Xylanases, xylanase families and extremophilic xilanases. FEMS Microbiol Rev 29:3–23PubMedGoogle Scholar
  6. 6.
    Csiszár E, Urbánszki K, Szakács G (2001) Biotreatment of desized cotton fabric by commercial cellulase and xylanase enzymes. J Mol Catalysis B: Enzymatic 11:1065–1072Google Scholar
  7. 7.
    Ferreira HM, Filho EXF (2004) Purification and characterization of a β-mannanase from Trichoderma harzianum strain T4. Carbohydr Polym 57:23–29Google Scholar
  8. 8.
    Filho EXF, Puls J, Coughlan MP (1993) Biochemical characteristics of two endo-β-1,4-xylanases produced by Penicillium capsulatum. J Ind Microbiol Biotechnol 11:171–180Google Scholar
  9. 9.
    Filho EXF, Puls J, Coughlan MP (1993) Physicochemical and catalytic properties of a low-molecular-weight endo-1,4-β-d-xylanase from Myrothecium verrucaria. Enzyme Microb Technol 15:535–540Google Scholar
  10. 10.
    Gawande PV, Kamat MY (1999) Production of Aspergillus xilanases by lignocellulosic waste fermentation and its application. J Appl Microbiol 87:511–519PubMedGoogle Scholar
  11. 11.
    Grabski AC, Jeffries TW (1991) Production, purification, and characterization of β-(1,4)-endoxylanase of Streptomyces roseiscleroticus. Appl Environ Microbiol 57:987–992PubMedGoogle Scholar
  12. 12.
    Haltrich D, Nidetzky B, Kulbe KD, Steiner W, Zupancic S (1996) Production of fungal xylanases. Biores Technol 58:137–161Google Scholar
  13. 13.
    Imoto T, Yamada H (1990) Chemical modification. In: Creighton TE (ed) Protein function, a practical approach. IRL Press, Oxford, pp 247–278Google Scholar
  14. 14.
    Ishihara M, Tawata S, Toyama S (1997) Purification and some properties of a thermostable xylanase from thermophilic fungus strain HG-1. J Ferment Bioeng 83:478–480Google Scholar
  15. 15.
    Ito K, Ogassawara J, Sugimoto T, Ishikawa T (1992) Purification and properties of acid stable xylanases form Aspergillus kawachii. Biosc Biotechnol Biochem 56:547–550Google Scholar
  16. 16.
    Jovanovic I, Magnuson JK, Collart F, Robbertse B, Adney WS, Himmel ME et al (2009) Fungal glycoside hydrolases for saccharification of lignocellulose: outlook for new discoveries fueled by genomics and functional studies. Cellulose 16:687–697Google Scholar
  17. 17.
    Kadam KL (2002) Environmental benefits on a life cycle basis of using bagasse-derived ethanol as a gasoline oxygenate in India. Energy Policy 30:371–384Google Scholar
  18. 18.
    Kapoor M, Singh A, Kuhad RC (2007) Application of xylanases in the pulp and paper industry: an appraisal. In: Kuhad RC, Singh A (eds) Lignocellulose biotechnology: future prospects. IK International, New Delhi, pp 307–310Google Scholar
  19. 19.
    Khan MA, Ashraf SM, Malhotra VP (2004) Development and characterization of a wood adhesive using bagasse lignin. Int J Adhesion and Adhesives 24:485–493Google Scholar
  20. 20.
    Krengel U, Dijkstra BW (1996) Three-dimensional structure of endo-1,4-β-xylanase I from Aspergillus niger: molecular basis for its low pH optimum. J Mol Biol 263:70–78PubMedGoogle Scholar
  21. 21.
    Kulkarni N, Shendye A, Rao M (1999) Molecular and biotechnological aspects of xilanases. FEMS Microbiol Rev 23:411–456PubMedGoogle Scholar
  22. 22.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedGoogle Scholar
  23. 23.
    Laxmi GS, Sathish T, Rao CS, Brahmaiah P, Hymavathi M, Prakasham RS (2008) Palm fiber as novel substrate for enhanced xylanase production by isolated Aspergillus sp. RSP-6. Curr Trends Biotechnol Pharm 2:447–455Google Scholar
  24. 24.
    Leatherbarrow RJ (1999) Enzfitter Manual, a non-linear curve fitting program for Windows. Biosoft, London, pp 1–104Google Scholar
  25. 25.
    Mandels M, Andreotii R, Roche C (1976) Measurement of saccharifying cellulase. Biotechnol Bioeng Symposium 16:21–33Google Scholar
  26. 26.
    Medeiros RG, Hanada R, Filho EXF (2003) Production of xylan-degrading enzymes from Amazon Forest fungal species. Int Biodet Biodegr 52:97–100Google Scholar
  27. 27.
    Medeiros RG, da Silva Jr FG, Báo SN, Hanada R, Filho EXF (2007) Application of xylanases from Amazon forest fungal species in bleaching of eucalyptus kraft pulps. Braz Arch Biol Technol 50:231–238Google Scholar
  28. 28.
    Miller G (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428Google Scholar
  29. 29.
    Paba J, Santana JM, Teixeira ARL, Fontes W, Sousa MV, Ricart CAO (2004) Proteomic analysis of the human pathogen Trypanosoma cruzi. Proteomics 4:1052–1059PubMedGoogle Scholar
  30. 30.
    Pandey A, Selvakumar P, Soccol CR, Nigam P (1999) Solid state fermentation for the production of industrial enzymes. Curr Sci 77:149–162Google Scholar
  31. 31.
    Parkkinen T, Hakulinen N, Tenkanen M, Siika-aho M, Rouvinen J (2004) Crystallization and preliminary X-ray analysis of a novel Trichoderma reesei xylanase IV belonging to glycoside hydrolase family 5. Acta Cryst Section D 60:542–544Google Scholar
  32. 32.
    Polizeli MLM, Rizzatti ACS, Monti R, Terenzi HF, Jorge JA, Amorim DS (2005) Xylanases from fungi: properties and industrial applications. Appl Microbiol Biotechnol 67:577–591PubMedGoogle Scholar
  33. 33.
    Raj HG, Saxena M, Allameh A (1992) Metabolism of foreign compounds by fungi. In: Arora DK, Elander RP, Mukerji KG (eds) Handbook of applied mycology. Marcel Dekker, New York, pp 881–904Google Scholar
  34. 34.
    Salama MA, Ismail KMI, Amany HA, El-Lill A, Geweely NSI (2008) Biochemical studies of purified extracellular xilanases from Aspergillus versicolor. Int J Bot 4:41–48Google Scholar
  35. 35.
    Schuster E, Dunn-Coleman N, Frisvad JC, van Dijck PW (2002) On the safety of Aspergillus niger—a review. Appl Microbiol Biotechnol 59:426–435PubMedGoogle Scholar
  36. 36.
    Shei JC, Fratzke AR, Frederick MM, Frederick JR, Reilly PJ (1985) Purification and characterization of endo-xylanases from Aspergillus niger. II. An enzyme of pI 4.5. Biotechnol Bioeng 27:533–538PubMedGoogle Scholar
  37. 37.
    Silva CHC, Puls J, Sousa MV, Filho EXF (1999) Purification and characterization of a low molecular weight xylanase from solid state cultures of Aspergillus fumigatus. Braz J Microbiol 30:114–119Google Scholar
  38. 38.
    Subramaniyan S, Prema P (2000) Cellulase-free xilanases from Bacillus and other microorganisms. FEMS Microbiol Lett 183:1–7PubMedGoogle Scholar
  39. 39.
    Subramaniyan S, Prema P (2002) Biotechnology of microbial xilanases: enzymology, molecular biology and application. Crit Rev Biotechnol 22:33–46PubMedGoogle Scholar
  40. 40.
    Tan LUL, Mayers P, Saddler JN (1987) Purification and characterization of a thermostable xylanase from thermophilic fungus Thermoascus aurantiacus. Can J Microbiol 33:689–692Google Scholar
  41. 41.
    Taneja K, Gupta S, Kuhad RC (2002) Properties and application of a partially purified alkaline xylanase from an alkalophilic fungus Aspergillus nidulans KK-99. Biores Technol 85:39–42Google Scholar
  42. 42.
    Teixeira RSS, Siqueira FG, Souza MV, Filho EXF, Bon EPS (2010) Purification and characterization studies of a thermostable β-xylanase from Aspergillus awamori. J Ind Microbiol Biotechnol 37:1041–1051PubMedGoogle Scholar
  43. 43.
    Wang P, Mason C, Broda P (1993) Xylanases from Streptomyces cyaneus: their production, purification and characterization. J Gen Microbiol 139:1987–1993Google Scholar
  44. 44.
    Wong KKY, Tan LUL, Saddler JN (1988) Multiplicity of β-1,4-xylanase in microorganisms: functions and applications. Microbiol Rev 52:305–317PubMedGoogle Scholar
  45. 45.
    Ximenes FA, Silveira FQP, Filho EXF (1996) Production of β-xylosidase activity by Trichoderma harzianum strains. Curr Microbiol 33:71–77Google Scholar
  46. 46.
    Ximenes FA, Sousa MV, Puls J, da Silva Jr FG, Filho EXF (1999) Purification and characterization of a low-molecular-weight xylanase produced by Acrophialophora nainiana. Curr Microbiol 38:18–21PubMedGoogle Scholar
  47. 47.
    Zhang YHP, Himmel ME, Mielenz JR (2006) Outlook for cellulase improvement: screening and selection strategies. Biotechnol Adv 24:452–481Google Scholar
  48. 48.
    Zhao J, Li X, Qu Y, Gao P (2002) Xylanase pretreatment leads to enhanced soda pulping of wheat straw. Enz Microb Tehnol 30:734–740Google Scholar

Copyright information

© Springer Science+Business Media, LLC. 2011

Authors and Affiliations

  • Natália von Gal Milanezi
    • 1
  • Diana Paola Gómez Mendoza
    • 2
  • Félix Gonçalves de Siqueira
    • 1
  • Luciano Paulino Silva
    • 3
  • Carlos André Ornelas Ricart
    • 2
  • Edivaldo Ximenes Ferreira Filho
    • 1
  1. 1.Laboratory of Enzymology, Department of Cellular BiologyUniversity of BrasíliaBrasíliaBrazil
  2. 2.Laboratory of Biochemistry and Protein Chemistry, Department of Cellular BiologyUniversity of BrasíliaBrasíliaBrazil
  3. 3.Laboratory of Mass Spectrometry, Embrapa Genetic Resources and BiotechnologyBrasíliaBrazil

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