Endo-1,4-β-xylanase B from Aspergillus cf. niger BCC14405 Isolated in Thailand: Purification, Characterization and Gene Isolation

  • Krisana, Asano ;
  • Rutchadaporng, Sriprang ;
  • Jarupan, Gobsuk ;
  • Lily, Eurwilaichitr ;
  • Sutipa, Tanapongpipat ;
  • Kanyawim, Kirtikara
  • Published : 2005.01.31


During the screening of xylanolytic enzymes from locally isolated fungi, one strain BCC14405, exhibited high enzyme activity with thermostability. This fugal strain was identified as Aspergillus cf. niger based on its morphological characteristics and internal transcribed spacer (ITS) sequences. An enzyme with xylanolytic activity from BCC14405 was later purified and characterized. It was found to have a molecular mass of ca. 21 kDa, an optimal pH of 5.0, and an optimal temperature of $55^{\circ}C$. When tested using xylan from birchwood, it showed $K_m$ and $V_{max}$ values of 8.9 mg/ml and 11,100 U/mg, respectively. The enzyme was inhibited by $CuSO_4$, EDTA, and by $FeSO_4$. The homology of the 20-residue N-terminal protein sequence showed that the enzyme was an endo-1,4-$\beta$-xylanase. The full-length gene encoding endo-1,4-$\beta$-xylanase from BCC14405 was obtained by PCR amplification of its cDNA. The gene contained an open reading frame of 678 bp, encoding a 225 amino acid protein, which was identical to the endo-1,4-$\^{a}$-xylanase B previously identified in A. niger.


Aspergillus cf. niger;Endo-1,4-$\beta$-xylanase B;Hemicellulase;Thermostability;Xylanase


  1. Bruins, M. E., Jasen, A. E. and Boom R. M. (2001) Thermozymes and their applications: a review of recent literature and patents. Appl. Biochem. Biotechnol. 90, 155-186
  2. Eriksson, K. E. L., Blanchette, R. A. and Ander, P. (1990) Biodegradation of hemicelluloses; in Microbial and Enzymatic Degradation of Wood and Wood Components, Eriksson, K. E. (ed.), pp. 181-187, Springer-Verlag, Berlin, Germany
  3. Gorbacheva, I. V. and Rodionova, N. A. (1977) Studies on xylan degrading enzymes. I. Purification and characterization of endo- 1,4-beta-xylanase from Aspergillus niger str. 14. Biochim. Biophys. Acta 484, 79-93
  4. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680- 685
  5. Sambrook, J. and Russell, D. W. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, New York, USA
  6. Varga, J., Rigo, K., Toth, B., Teren, J. and Kozakiewicz, Z. (2003) Evolutionary relationships among Aspergillus species producing economically important mycotoxins. Food Technol. Biotechnol. 41, 29-36
  7. Velegraki, A., Kambouris, M., Kostourou, A., Chalevelakis, G. and Legakis, N. J. (1999) Rapid extraction of fungal DNA from clinical samples for PCR amplification. Food Technol. Biotechnol. 37, 69-73
  8. Yeates, C., Gillings, M. R., Davison, A. D., Altavilla, N. and Veal, D. A. (1998) Methods for microbial DNA extraction from soil for PCR amplification. Biol. Proced. Online 1, 40-47
  9. Hawksworth, D. L. (2001) The Magnitude of fungal diversity: the 1.5 million species estimated revisited. Mycol. Res. 105, 1422- 1432
  10. Tahir, T. A., Berrin, J. G., Flatman, R., Roussel, A., Roepstorff, P., Williamson, G. and Juge, N. (2002) Specific characterization of substrate and inhibitor binding sites of a glycosyl hydrolase family 11 xylanase from Aspergillus niger. J. Biol. Chem. 277, 44035-44043
  11. Singh, S., Madlala, A. M. and Prior, B. A. (2003) Thermomyces lanuginosus: properties of strains and their hemicellulases. FEMS Microbiol. Rev. 27, 3-16
  12. Buchan, A., Newell, S. Y., Moreta, J. I. and Moran, M. A. (2002) Analysis of internal transcribed spacer (ITS) regions of rRNA genes in fungal communities in a southeastern U.S. salt marsh. Microb. Ecol. 43, 329-340
  13. George, S. P., Ahmad, A. and Rao, M. B. (2001) A novel thermostable xylanase from Thermomonospora sp.: influence of additives on thermostability. Bioresour. Technol. 78, 221-224
  14. Berrin, J. G., Williamson, G., Puigserver, A., Chaix, J. C., McLauchlan, W. R. and Juge, N. (2000) High-level production of recombinant fungal endo-beta-1,4-xylanase in the methylotrophic yeast Pichia pastoris. Protein Expr. Purif. 19, 179-187
  15. Lin, J., Ndlovu, L. M., Singh, S. and Pillay, B. (1999) Purification and biochemical characteristics of beta-D-xylanase from a thermophilic fungus, Thermomyces lanuginosus-SSBP. Biotechnol. Appl. Biochem. 30, 73-79
  16. Sunna, A. and Antranikian, G. (1997) Xylanolytic enzymes from fungi and bacteria. Crit. Rev. Biotechnol. 17, 39-67
  17. Bailey, M. J., Biely, P. and Poutanen, K. (1992) Interlaboratory testing of methods for assay of xylanase activity. J. Biotechnol. 23, 257-270
  18. John, M., Schmidt, B. and Schmidt, J. (1979) Purification and some properties of five endo-1,4-beta-D-xylanases and a beta- D-xylosidase produced by a strain of Aspergillus niger. Can. J. Biochem. 57,125-134
  19. Taneja, K., Gupta, S. and Kuhad, R. C. (2002) Properties and application of a partially purified alkaline xylanase from an alkalophilic fungus Aspergillus nidulans KK-99. Bioresour. Technol. 85, 39-42
  20. Wong, K. K. Y., Tan, L. U. L. and Saddler, J. N. (1988) Multiplicity of $\beta$-1,4-xylanase in microorganisms: function and applicants. Microbiol. Rev. 52, 305-317
  21. Biely, P. (1993) Biochemical aspects of the microbial hemicellulases; in Hemicelluloses and Hemicellulase, Coughlan, M. and Hazlewood, G. (eds.), pp. 29-51, Portland Press, London, UK
  22. Plumb, J. A., Milroy, R. and Kaye, S. B. (1989) Effects of the pH dependence of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetazolium bromide-formazan absorption on chemosensitivity determined by a novel tetrazolium-based assay. Cancer Res. 49, 4435- 4440
  23. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254

Cited by

  1. Improvement of thermostability of fungal xylanase by using site-directed mutagenesis vol.126, pp.4, 2006,
  2. Biochemical characterization, cloning and molecular modeling of a detergent and organic solvent-stable family 11 xylanase from the newly isolated Aspergillus niger US368 strain vol.47, pp.12, 2012,
  3. Molecular cloning and heterologous expression of a new xylanase gene from Plectosphaerella cucumerina vol.74, pp.2, 2007,
  4. Biochemical and Biophysical Characterization of Purified Thermophilic Xylanase Isoforms in Cereus pterogonus Plant Spp vol.31, pp.2, 2012,
  5. Molecular characterization of a glycosyl hydrolase family 10 xylanase from Aspergillus niger vol.92, pp.2, 2013,
  6. Production and immobilization of enzymes by solid-state fermentation of agroindustrial waste vol.38, pp.3, 2015,
  7. Purification and Characterization of Two Extracellular Xylanases from Penicillium sclerotiorum: A Novel Acidophilic Xylanase vol.162, pp.2, 2010,
  8. Identification of three important amino acid residues of xylanase AfxynA from Aspergillus fumigatus for enzyme activity and formation of xylobiose as the major product vol.50, pp.4, 2015,
  9. A novel halotolerant xylanase from marine isolate Bacillus subtilis cho40: gene cloning and sequencing vol.28, pp.6, 2011,
  10. Analysis of functional xylanases in xylan degradation by Aspergillus niger E-1 and characterization of the GH family 10 xylanase XynVII vol.2, pp.1, 2013,
  11. GH11 xylanases: Structure/function/properties relationships and applications vol.30, pp.3, 2012,
  12. Purification, characterization and mass spectroscopic analysis of thermo-alkalotolerant β-1, 4 endoxylanase from Bacillus sp. and its potential for dye decolorization vol.65, pp.2, 2011,
  13. Molecular cloning and characterization of a GH11 endoxylanase from Chaetomium globosum, and its use in enzymatic pretreatment of biomass vol.97, pp.16, 2013,
  14. Gene cloning, expression and characterization of a new cold-active and salt-tolerant endo-β-1,4-xylanase from marine Glaciecola mesophila KMM 241 vol.84, pp.6, 2009,
  15. Purification and Characterization of a Liver-derived β-N-Acetylhexosaminidase from Marine Mammal Sotalia fluviatilis vol.29, pp.3, 2010,
  16. A thermotolerant β-glucosidase isolated from an endophytic fungi, Periconia sp., with a possible use for biomass conversion to sugars vol.67, pp.2, 2009,
  17. Optimization of the Trichoderma reesei endo-1,4-beta-xylanase production by recombinant Pichia pastoris vol.52, pp.1, 2010,
  18. Purification and characterization of a thermostable xylanase from the brown-rot fungus Laetiporus sulphureus vol.107, pp.1, 2009,
  19. Site-Directed Mutagenesis and Thermostability of Xylanase XYNB from Aspergillus niger 400264 vol.62, pp.1, 2011,
  20. Purification and Properties of a Thermostable Xylanase GH 11 from Penicillium occitanis Pol6 vol.168, pp.4, 2012,
  21. Purification and Characterization of Thermophilic Xylanase Isolated from the Xerophytic-Cereus pterogonus sp. vol.29, pp.7, 2010,
  22. Biodeinking of old newspaper pulp using a cellulase-free xylanase preparation of Aspergillus niger DX-23 vol.5, 2016,
  23. Cloning, expression, and characterization of a xylanase 10 from Aspergillus terreus (BCC129) in Pichia pastoris vol.46, pp.1, 2006,
  24. Cloning and expression of xylanase 10 fromCryptovalsa mangrovei(BCC7197) inPichia pastoris vol.16, pp.5, 2005,
  25. Purification and biochemical characterization of two major thermophilic xylanase isoforms (T70 and T90) from xerophytic Opuntia vulgaris plant spp. vol.19, pp.4, 2012,
  26. Xylan decomposition by Aspergillus clavatus endo-xylanase vol.68, pp.1, 2009,
  27. Thermostability improvement of Orpinomyces sp. xylanase by directed evolution vol.81, 2012,
  28. Decomposition of sugarcane bagasse with lignocellulose-derived thermotolerant and thermoresistant Penicillia and Aspergilli vol.92, 2014,
  29. Molecular cloning of fungal xylanases: an overview vol.84, pp.1, 2009,
  30. Purification of xylanase from Aspergillus niger DFR-5: Individual and interactive effect of temperature and pH on its stability vol.46, pp.4, 2011,
  31. Mapping the polysaccharide degradation potential of Aspergillus niger vol.13, pp.1, 2012,
  32. Cloning, expression, characterization, and high cell-density production of recombinant endo-1,4-β-xylanase from Aspergillus niger in Pichia pastoris vol.41, pp.1-2, 2007,
  33. Cloning and characterization of xylanase in cellulolytic Bacillus sp. strain JMY1 isolated from forest soil vol.59, pp.3, 2016,
  34. Biochemical characterization and kinetic comparison of encapsulated haze removing acidophilic xylanase with partially purified free xylanase isolated from Aspergillus flavus MTCC 9390 vol.52, pp.1, 2015,
  35. Molecular Identification Using ITS Sequences and Genome Shuffling to Improve 2-Deoxyglucose Tolerance and Xylanase Activity of Marine-Derived Fungus, Aspergillus Sp. NRCF5 vol.167, pp.8, 2012,
  36. A Highly Efficient Xylan-Utilization System in Aspergillus niger An76: A Functional-Proteomics Study vol.9, pp.1664-302X, 2018,