Skip to main content
SpringerLink
Log in

Purification and Physicochemical Characterization of a Novel Thermostable Xylanase Secreted by the Fungus Myceliophthora heterothallica F.2.1.4

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

Xylanases are enzymes that act in the depolymerization of xylan and that can be used in the food industry, the paper industry, and for bioenergy, among other uses. In this context, particular emphasis is devoted to xylooligosaccharides (XOS) that act as prebiotics, which, under the action of probiotic microorganisms, are capable of positively modifying the intestinal microbiota. In this sense, searching for microbial xylanases stands out as a sustainable strategy for the production of prebiotics. To date, there have been no reports in the literature regarding the purification of native xylanase from Myceliophthora heterothallica F.2.1.4. In this study, a xylanase from this fungus was purified and characterized. The xylanase, with 27 kDa, showed maximum activity at pH 4.5 and 65–70 °C. It maintained more than 80% of its residual activity when exposed to (i) temperatures between 30 and 60 °C for 1 h and (ii) pH 5–10 for 24 h at 4 and 25 °C. These high tolerances to different pH and different temperatures are important properties that add value to this enzyme. The hydrolysates of this enzyme on beechwood xylan, analyzed by HPAE-PAD, were mostly xylobiose (X2) and xylotriose (X3). Hydrolysates were also quantified, being retrieved from 234.2 mg xylooligosaccharides/g of hydrolyzed xylan for 12 h. According to the products obtained from the xylan hydrolysis and its tolerance properties of the enzyme, it has demonstrated potential for application production of xylooligosaccharides for use as prebiotics.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. CAZy - Carbohydrate Active Enzymes database. Available from: www.cazy.org. Acessed June 26, 2018.

  2. Polizeli, M. L. T. M., Rizzatti, A. C. S., Monti, R., Terenzi, H. F., Jorge, J. A., & Amorim, D. S. (2005). Xylanases from fungi: properties and industrial applications. Applied Microbiology and Biotechnology, 67(5), 577–591.

    Article  CAS  Google Scholar 

  3. Verma, D., Anand, A., & Satyanarayana, T. (2013). Thermostable and alkalistable endoxylanase of the extremely thermophilic bacterium Geobacillus thermodenitrificans TSAA1: cloning, expression, characteristics and its applicability in generating xylooligosaccharides and fermentable sugars. Applied Biochemistry and Biotechnology, 170(1), 119–130.

    Article  CAS  Google Scholar 

  4. Liu, X., Liu, Y., Jiang, Z., Liu, H., Yang, S., & Yan, Q. (2018). Biochemical characterization of a novel xylanase from Paenibacillus barengoltzii and its application in xylooligosaccharides production from corncobs. Food Chemistry, 264, 310–318.

    Article  CAS  Google Scholar 

  5. Gibson, G. R., Hutkins, R., Sanders, M. E., Prescott, S. L., Reimer, R. A., Salminen, S. J., Scott, K., Stanton, C., Swanson, K. S., Cani, P. D., Verbeke, K., & Reid, G. (2017). The International Scientific Association for Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of prebiotics. Nature Reviews. Gastroenterology & Hepatology, 14, 491–502.

    Article  Google Scholar 

  6. Vrese, M. & Schrezenmeir, J. (2008). Probiotics, prebiotics, and synbiotics. Advances in Biochemical Engineering/Biotechnology, 111, 1–66.6.

  7. Akpinar, O., Erdogan, K., & Bostanci, S. (2009). Enzymatic production of xylooligosaccharide from selected agricultural wastes. Food and Bioproducts Processing, 87(2), 145–151.

    Article  CAS  Google Scholar 

  8. Vázquez, M. J., Alonso, J. L., Domínguez, H., & Parajó, J. C. (2002). Enzymatic processing of crude xylooligomer solutions obtained by autohydrolysis of eucalyptus wood. Food Biotechonology, 16, 91–105.

    Article  Google Scholar 

  9. Brienzo, M., Carvalho, W., & Milagres, A. M. F. (2010). Xylooligosaccharides production from alkali-pretreated sugarcane bagasse using xylanases from Thermoascus aurantiacus. Applied Biochemistry and Biotechnology, 162(4), 1195–1205.

    Article  CAS  Google Scholar 

  10. Bajpai, P (2014). Xylanolytic enzymes. Chapter 6 - purification of xylanases, Academic Press, 53–61.

  11. Bhalla, A., Bansal, N., Kumar, S., Bischoff, K. M., & Sani, R. K. (2013). Improved lignocellulose conversion to biofuels with thermophilic bacteria and thermostable enzymes. Bioresource Technology, 128, 751–759.

    Article  CAS  Google Scholar 

  12. Chapla, D., Pandit, P., & Shah, A. (2012). Production of xylooligosaccharides from corncob xylan by fungal xylanase and their utilization by prebiotics. Bioresource Technology, 115, 215–221.

    Article  CAS  Google Scholar 

  13. Moretti, M. M. S., Bocchini-Martins, D. A., Silva, R., Rodrigues, A., Sette, L. D., & Gomes, E. (2012). Selection of thermophilic and thermotolerant fungi for the production of cellulases and xylanases under solid-state fermentation. Brazilian Journal of Microbiology, 43(3), 1062–1071.

    Article  CAS  Google Scholar 

  14. Silva, V. C. T.; Coto, A. L. S.; Souza, R. C.; Neves, M. B. S.; Gomes, E. & Bonilla-Rodriguez, G. O (2016). Effect of pH, temperature, and chemicals on the endoglucanases and 훽-glucosidases from the thermophilic fungus Myceliophthora heterothallica F.2.1.4. Obtained by solid-state and submerged cultivation. Biochemistry Research International, 2016, 1–9.

  15. Diaz, A. B., Moretti, M. M. S., Bezerra-Bussoli, C., Nunes, C. C. C., Blandino, A., Silva, R., & Gomes, E. (2015). Evaluation of microwave-assisted pretreatment of lignocellulosic biomass immersed in alkaline glycerol for fermentable sugars production. Bioresource Technology, 185, 316–323.

    Article  CAS  Google Scholar 

  16. Zanphorlin, L. M., Facchini, F. D. A., Vasconcelos, F., Bonugli-Santos, R. C., Rodrigues, A., Sette, L. D., Gomes, E., & Bonilla-Rodriguez, G. O. (2010). Production, partial characterization, and immobilization in alginate beads of an alkaline protease from a new thermophilic fungus Myceliophthora sp. The Journal of Microbiology, 48(3), 331–336.

    Article  CAS  Google Scholar 

  17. Bailey, M. J., Biely, P., & Poutanen, K. (1992). Interlaboratory testing of methods for assay of xylanase activity. Journal of Biotechnology, 23, 257–270.

    Article  CAS  Google Scholar 

  18. Miller, G. L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31(3), 426–429.

    Article  CAS  Google Scholar 

  19. See, Y. S., & Jackowski, G. (1989). Estimating molecular weights of polypeptides by SDS gel electrophoresis. In T. E. Creigton (Ed.), Protein structure a practical approach (pp. 1–19). New York: Oxford University.

    Google Scholar 

  20. Silva, R. R., Caetano, R. C., Okamoto, D. N., Oliveira, L. C. G., Bertolin, T. C., Juliano, M. A., Juliano, L., Oliveira, A. H. C., Rosa, J. C., & Cabral, H. (2014). The identification and biochemical properties of the catalytic specificity of a serine peptidase secreted by Aspergillus fumigatus Fresenius. Protein and Peptide Letters, 21(7), 663–671.

    Article  Google Scholar 

  21. Silva, R. R., Oliveira, L. C. G., Juliano, M. A., Juliano, L., Oliveira, A. H. C., Rosa, J. C., & Cabral, H. (2017a). Biochemical and milk-clotting properties and mapping of catalytic subsites of an extracellular aspartic peptidase from basidiomycete fungus Phanerochaete chrysosporium. Food Chemistry, 225, 45–54.

    Article  Google Scholar 

  22. Silva, R. R., Oliveira, L. C. G., Juliano, M. A., Juliano, L., Rosa, J. C., & Cabral, H. (2017b). Activity of a peptidase secreted by Phanerochaete chrysosporium depends on lysine to subsite S’1. International Journal of Biological Macromolecules, 94, 474–483.

    Article  Google Scholar 

  23. Noble, J. E. & Bailey, M. J. A. (2009). Quantification of protein. Methods in enzymology, pp. 81. In: Lorsch, J. (Ed.), Methods in enzymology. Academic Press, 463, 73–95.

  24. Noble, J. E. (2014): Chapter two-quantification of protein concentration using UV absorbance and coomassie dyes, pp. 21. In: Lorsch, J. (Ed.), Methods in enzymology, Academic Press, 536.

  25. McPhillips, K., Waters, D. M., Parlet, C., Walsh, D. J., Arendt, E. K., & Murray, P. G. (2014). Purification and characterisation of a β-1,4-xylanase from Remersonia thermophila CBS 540.69 and its application in bread making. Applied Biochemistry and Biotechnology, 172(4), 1747–1762.

    Article  CAS  Google Scholar 

  26. Beaugrand, J., Chambat, G., Wong, V. W. K., Goubet, F., Rémond, C., Paës, G., Benamrouche, S., Debeire, P., O’Donohue, M., & Chabbert, B. (2004). Impact and efficiency of GH10 and GH11 thermostable endoxylanases on wheat bran and alkali-extractable arabinoxylans. Carbohydrate Research, 339(15), 2529–2540.

    Article  CAS  Google Scholar 

  27. Sharma, M., Chadha, B. S., & Saini, H. S. (2010). Purification and characterization of two thermostable xylanases from Malbranchea flava active under alkaline conditions. Bioresource Technology, 101(22), 8834–8842.

    Article  CAS  Google Scholar 

  28. Fawzi, E. M. (2011). Highly thermostable xylanase purified from Rhizomucor miehei NRL 3169. Acta Biologica Hungarica, 62(1), 85–94.

    Article  CAS  Google Scholar 

  29. Basit, A., Liu, J., Miao, T., Zheng, F., Rahim, K., Lou, H., & Jiang, W. (2018). Characterization of two endo-β-1,4-xylanases from Myceliophthora thermophila and their saccharification efficiencies, synergistic with commercial cellulase. Frontiers in Microbiology, 9(233), 1–11.

    Google Scholar 

  30. Souza, A. R., Araújo, G. C., Zanphorlin, L. M., Ruller, R., Franco, F. C., Torres, F. A. G., Mertens, J. A., Bowman, M. J., Gomes, E., & Silva, R. (2016). Engineering increased thermostability in the GH-10 endo-1,4-β-xylanase from Thermoascus aurantiacus CBMAI 756. International Journal of Biological Macromolecules, 93(Pt A), 20–26.

    Article  Google Scholar 

  31. Mosier, N., Wyman, C., Dale, B., Elander, R., Lee, Y. Y., Holtzapple, M., & Ladisch, M. (2005). Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresource Technology, 96(6), 673–686.

    Article  CAS  Google Scholar 

  32. Turunen, O., Etuaho, K., Fenel, F., Vehmaanpera, J., Wu, X., Rouvinen, J., & Leisola, M. (2001). A combination of weakly stabilizing mutations with a disulfide bridge in the α-helix region of Trichoderma reesei endo-1,4-β-xylanase II increases the thermal stability through synergism. Journal of Biotechnology, 88(1), 37–46.

    Article  CAS  Google Scholar 

  33. Singh, B. (2014). Myceliophthora thermophila syn. Sporotrichum thermophile: a thermophilic mould of biotechnological potential. Critical Reviews in Biotechnology, 1–11.

  34. Kannan, A.; Hettiarachchy, N. S. & Marshall, M. R. (2012). Food proteins - peptides. In: Hettiarachchy, N. S. et al. Food proteins and peptides chemistry, functionality, interactions, and commercialization, CRC Press, 1–24.

  35. Al-Darkazali, H., Meevootisom, V., Isarangkul, D., & Wiyakrutta, S. (2017). Gene expression and molecular characterization of a xylanase from chicken cecum metagenome. International Journal of Microbiology, 2017, 1–12.

    Article  Google Scholar 

  36. Zhao, H. (2005). Effect of ions and other compatible solutes on enzyme activity, and its implication for biocatalysis using ionic liquids. Review. Journal of Molecular Catalysis B Enzymatic, 37, 16–25, 1-6.

  37. Mizrahi, L., & Achituv, Y. (1989). Effect of heavy metals ions on enzyme activity in the mediterranean mussel, Donax trunculus. Bulletin of Environmental Contamination and Toxicology, 42(6), 854–859.

    Article  CAS  Google Scholar 

  38. Ding, C., Li, M., & Hu, Y. (2018). High-activity production of xylanase by Pichia stipitis: purification, characterization, kinetic evaluation and xylooligosaccharides production. Biological Macromolecules, 117, 72–77.

    Article  CAS  Google Scholar 

  39. Michelin, M., Silva, T. M., Jorge, J. A., & Polizeli, M. L. T. M. (2014). Purification and biochemical properties of multiple xylanases from Aspergillus ochraceus tolerant to Hg2+ ion and a wide range of pH. Applied Biochemistry and Biotechnology, 174(1), 206–220.

    Article  CAS  Google Scholar 

  40. Harris, A. D., & Ramalingam, C. (2010). Xylanases and its application in food industry: a review. Journal of Experimental Sciences, 1, 10–11.

    Google Scholar 

  41. Juturu, V., & Wu, J. C. (2012). Microbial xylanases: engineering, production and industrial applications. Biotechnology Advances, 30(6), 1219–1227.

    Article  CAS  Google Scholar 

  42. Yang, H., Wang, K., Song, X., & Xu, F. (2011). Production of xylooligosaccharides by xylanase from Pichia stipitis based on xylan preparation from triploid Populas tomentosa. Bioresource Technology, 102(14), 7171–7176.

    Article  CAS  Google Scholar 

  43. Otieno, D. O., & Ahring, B. K. (2012). The potential for oligosaccharide production from the hemicellulose fraction of biomasses through pretreatment processes: xylooligosaccharides (XOS), arabinooligosaccharides (AOS), and mannooligosaccharides (MOS). Carbohydrate Research, 360, 84–92.

    Article  CAS  Google Scholar 

  44. Bragatto, J., Segato, F., & Squina, F. M. (2013). Production of xylooligosaccharides (XOS) from delignified sugarcane bagasse by peroxide-HAc process using recombinant xylanase from Bacillus subtilis. Industrial Crops and Products, 51, 123–129.

    Article  CAS  Google Scholar 

  45. Samanta, A. K., Jayapal, N., Jayaram, C., Roy, S., Kolte, A. P., Senani, S., & Sridhar, M. (2015). Xylooligosaccharides as prebiotics from agricultural by-products: production and applications. Bioactive Carbohydrates and Dietary Fibre, 5(1), 62–71.

    Article  CAS  Google Scholar 

  46. Linares-Pasten, J. A., Aronsson, A., & Karlsson, E. N. (2018). Structural considerations on the use of endo-xylanases for the production of prebiotic xylooligosaccharides from biomass. Current Protein and Peptide Science, 19(1), 48–67.

    CAS  PubMed  Google Scholar 

  47. Hoffmam, Z. B., Zanphorlin, L. M., Cota, J., Diogo, J. A., Almeida, G. B., Damásio, A. R. L., Squina, F., Murakami, M. T., & Ruller, R. (2016). Xylan-specific carbohydrate-binding module belonging to family 6 enhances the catalytic performance of a GH11 endo-xylanase. New Biotechnology, 33(4), 467–472.

    Article  CAS  Google Scholar 

  48. Santos, C. R., Hoffmam, Z. B., Martins, V. P. M., Zanphorlin, L. M., Assis, L. H. P., Honorato, R. V., Oliveira, P. S. L., Ruller, R., & Murakami, M. T. (2014). Molecular mechanisms associated with xylan degradation by xanthomonas plant pathogens. The Journal of Biological Chemistry, 289(46), 32186–32200.

    Article  CAS  Google Scholar 

  49. Gullón, P., González-Muñoz, M. J., & Parajó, J. C. (2011). Manufacture and prebiotic potential of oligosaccharides derived from industrial solid wastes. Bioresource Technology, 102(10), 6112–6119.

    Article  Google Scholar 

  50. Silva, R. R. (2017). Bacterial and fungal proteolytic enzymes, production, catalysis and potential applications. Applied Biochemistry and Biotechnology, 183(1), 1–19.48.

    Article  Google Scholar 

  51. Silva, R. R., Pedezzi, R., & Souto, T. B. (2017). Exploring the bioprospecting and biotechnological potential of white-rot and anaerobic Neocallimastigomycota fungi: peptidases, esterases, and lignocellulolytic enzymes. Applied Microbiology and Biotechnology, 101(8), 3089–3101.

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Process 2017/16482-5, and Conselho de Desenvolvimento Científico e Tecnológico (CNPq), Process 426578/2016-3. M. B, E.G, and R.S are research fellows of CNPq. The authors would like to thank Dr. Célia Maria Landi Franco and Dr. Márcia Maria de Souza Moretti for their help with the HPLC analysis.

Author information

Authors and Affiliations

  1. São Paulo State University (Unesp), Institute of Biosciences, Humanities and Exact Sciences, São José do Rio Preto, São Paulo, Brazil

    Lorena Caixeta de Oliveira Simões, Ronivaldo Rodrigues da Silva, Carlos Eduardo de Oliveira Nascimento, Maurício Boscolo, Eleni Gomes & Roberto da Silva

Authors
  1. Lorena Caixeta de Oliveira Simões
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ronivaldo Rodrigues da Silva
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Carlos Eduardo de Oliveira Nascimento
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Maurício Boscolo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Eleni Gomes
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Roberto da Silva
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Roberto da Silva.

Ethics declarations

Conflict of Interest

The authors declare that they have no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

de Oliveira Simões, L.C., da Silva, R.R., de Oliveira Nascimento, C.E. et al. Purification and Physicochemical Characterization of a Novel Thermostable Xylanase Secreted by the Fungus Myceliophthora heterothallica F.2.1.4. Appl Biochem Biotechnol 188, 991–1008 (2019). https://doi.org/10.1007/s12010-019-02973-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-019-02973-8

Keywords

Search

Navigation