Advertisement

Molecular Biotechnology

, Volume 58, Issue 12, pp 821–831 | Cite as

Expression of Two Novel β-Glucosidases from Chaetomium atrobrunneum in Trichoderma reesei and Characterization of the Heterologous Protein Products

  • Ana C. ColabardiniEmail author
  • Mari Valkonen
  • Anne Huuskonen
  • Matti Siika-aho
  • Anu Koivula
  • Gustavo H. Goldman
  • Markku Saloheimo
Original Paper

Abstract

Two novel GH3 family thermostable β-glucosidases from the filamentous fungus Chaetomium atrobrunneum (CEL3a and CEL3b) were expressed in Trichoderma reesei, purified by two-step ion exchange chromatography, and characterized. Both enzymes were active over a wide range of pH as compared to Neurospora crassa β-glucosidase GH3-3, which was also expressed in T. reesei and purified. The optimum temperature of both C. atrobrunneum enzymes was around 60 °C at pH 5, and both enzymes had better thermal and pH stability and higher resistance to metallic compounds and to glucose inhibition than GH3-3. They also showed higher activity against oligosaccharides composed of glucose units and linked with β-1,4-glycosidic bonds and moreover, had higher affinity for cellotriose over cellobiose. In hydrolysis tests against Avicel cellulose and steam-exploded sugarcane bagasse, performed at 45 °C, particularly the CEL3a enzyme performed similarly to N. crassa GH3-3 β-glucosidase. Taking into account the thermal stability of the C. atrobrunneum β-glucosidases, they both represent promising alternatives as enzyme mixture components for improved cellulose saccharification at elevated temperatures.

Keywords

β-glucosidase Chaetomium atrobrunneum Thermostability Bioethanol Hydrolysis 

Notes

Acknowledgments

This work was supported by jointly by Finland and Brazil in sustainable energy (Academy of Finland-CNPq), decision number 271146 of Academy of Finland, National Council of Scientific and Technological Development (CNPq), Brazil (490249/2012-4 Bilateral agreements/Call number 30/2012 - CNPq/AKA FINLÂNDIA), and the Foundation for Research of São Paulo State (FAPESP), Brazil. The fungal genome sequencing was supported from the project ‘Metagenome’ funded by the Finnish Funding Agency for Innovation, decision number 40148/07. We thank Dr Merja Oja for sequence search, Dr Martina Andberg for assistance with the protein purification and for providing some of the substrates, and Dr. Teun Boekhout, The Fungal Biodiversity Centre (CBS), the Netherlands, for providing the C. atrobrunneum CBS 269.91 strain.

Supplementary material

12033_2016_9981_MOESM1_ESM.docx (18 kb)
Supplementary material 1 (DOCX 17 kb)
12033_2016_9981_MOESM2_ESM.pptx (69 kb)
Supplementary material 2 (PPTX 68 kb)

References

  1. 1.
    Ankudimova, N. V., Baraznenok, V. A., Becker, E. G., & Okunev, O. N. (1999). Cellulase complex from Chaetomium cellulolyticum: Isolation and properties of major components. Biochemistry, 64, 1068–1073.Google Scholar
  2. 2.
    Barron, M. A., Sutton, D. A., Veve, R., Guarro, J., Rinaldi, M., Thompson, E., et al. (2003). Invasive mycotic infections caused by Chaetomium perlucidum, a new agent of cerebral phaeohyphomycosis. Journal of Clinical Microbiology, 41, 5302–5307.CrossRefGoogle Scholar
  3. 3.
    Bhatia, Y., Mishra, S., & Bisaria, V. S. (2002). Microbial beta-glucosidases: cloning, properties, and applications. Critical Reviews in Biotechnology, 22, 375–407.CrossRefGoogle Scholar
  4. 4.
    Blumer-Schuette, S. E., Brown, S. D., Sander, K. B., Bayer, E. A., Kataeva, I., Zurawski, J. V., et al. (2014). Thermophilic lignocellulose deconstruction. FEMS Microbiology Reviews, 38, 393–448.CrossRefGoogle Scholar
  5. 5.
    Bohlin, C., Olsen, S. N., Morant, M. D., Patkar, S., Borch, K., & Westh, P. (2010). A comparative study of activity and apparent inhibition of fungal beta-glucosidases. Biotechnology and Bioengineering, 107, 943–952.CrossRefGoogle Scholar
  6. 6.
    Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254.CrossRefGoogle Scholar
  7. 7.
    Canilha, L., Chandel, A. K., Milessi, T. S. S., Antunes, F. A. F., Freitas, W. L. C., Felipe, M. G. A., et al. (2012). Bioconversion of sugarcane biomass into ethanol: An overview about composition, pretreatment methods, detoxification of hydrolysates, enzymatic saccharification, and ethanol fermentation. Journal of Biomedicine and Biotechnology. doi: 10.1155/2012/989572.Google Scholar
  8. 8.
    Cuomo, C. A., Untereiner, W. A., Ma, L. J., Grabherr, M., & Birren, B. W. (2015). Draft genome sequence of the cellulolytic fungus Chaetomium globosum. Genome Announcements. doi: 10.1128/genomeA.00021-15.Google Scholar
  9. 9.
    de Giuseppe, P. O., Souza, T. A., Souza, F. H., Zanphorlin, L. M., Machado, C. B., Ward, R. J., et al. (2014). Structural basis for glucose tolerance in GH1 beta-glucosidases. Acta Crystallographica. Section D, Biological Crystallography, 70, 1631–1639.CrossRefGoogle Scholar
  10. 10.
    El-Gindy, A. A., Saad, R. R., & Fawzi, E. (2003). Purification and some properties of exo-1,4-beta-glucanase from Chaetomium olivaceum. Acta Microbiologica Polonica, 52, 35–44.Google Scholar
  11. 11.
    Ghose, T. K. (1987). Measurement of cellulase activities. Pure and Applied Chemistry, 59, 257–268.Google Scholar
  12. 12.
    Gietz, R. D. (2014). Yeast transformation by the LiAc/SS carrier DNA/PEG method. Methods in Molecular Biology, 1163, 33–44.CrossRefGoogle Scholar
  13. 13.
    Häkkinen, M., Arvas, M., Oja, M., Aro, N., Penttilä, M., Saloheimo, M., et al. (2012). Re-annotation of the CAZy genes of Trichoderma reesei and transcription in the presence of lignocellulosic substrates. Microbial Cell Factories, 11, 134–160.CrossRefGoogle Scholar
  14. 14.
    Hodge, D. B., Karim, M. N., Schell, D. J., & McMillan, J. D. (2009). Model-based fed-batch for high-solids enzymatic cellulose hydrolysis. Applied Biochemistry and Biotechnology, 152, 88–107.CrossRefGoogle Scholar
  15. 15.
    Kim, I. J., Nam, K. H., Yun, E. J., Kim, S., Youn, H. J., Lee, H. J., et al. (2015). Optimization of synergism of a recombinant auxiliary activity 9 from Chaetomium globosum with cellulase in cellulose hydrolysis. Applied Microbiology and Biotechnology, 99, 8537–8547.CrossRefGoogle Scholar
  16. 16.
    Kuhad, R. C., Singh, A., & Eriksson, K. E. (1997). Microorganisms and enzymes involved in the degradation of plant fiber cell walls. Advances in Biochemical Engineering/Biotechnology, 57, 45–125.CrossRefGoogle Scholar
  17. 17.
    Kumar, R., Singh, S., & Singh, O. V. (2008). Bioconversion of lignocellulosic biomass: Biochemical and molecular perspectives. Journal of Industrial Microbiology and Biotechnology, 35, 377–391.CrossRefGoogle Scholar
  18. 18.
    McIlvaine, T. C. (1921). A buffer solution for colorimetric comparaison. Journal of Biological Chemistry, 49, 183–186.Google Scholar
  19. 19.
    Nevalainen, H., & Peterson, R. (2014). Making recombinant proteins in filamentous fungi-are we expecting too much? Front Microbiology, 5, 75.Google Scholar
  20. 20.
    Papageorgiou, A. C., & Li, D. (2015). Expression, purification and crystallization of a family 55 beta-1,3-glucanase from Chaetomium thermophilum. Acta Crystallographica Section F: Structural Biology Communications, 71, 680–683.Google Scholar
  21. 21.
    Penttilä, M., Nevalainen, H., Rättö, M., Salminen, E., & Knowles, J. (1987). A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei. Gene, 61, 155–164.CrossRefGoogle Scholar
  22. 22.
    Rahikainen, J., Moilanen, U., Nurmi-Rantala, S., Lappas, A., Koivula, A., Viikari, L., et al. (2013). Effect of temperature on lignin-derived inhibition studied with three structurally different cellobiohydrolases. Bioresource Technology, 146, 118–125.CrossRefGoogle Scholar
  23. 23.
    Saloheimo, M., & Pakula, T. M. (2012). The cargo and the transport system: Secreted proteins and protein secretion in Trichoderma reesei (Hypocrea jecorina). Microbiology, 158, 46–57.CrossRefGoogle Scholar
  24. 24.
    Sipos, B., Benko, Z., Dienes, D., Réczey, K., Viikari, L., & Siika-aho, M. (2012). Characterisation of specific activities and hydrolytic properties of cell-wall-degrading enzymes produced by Trichoderma reesei Rut C30 on different carbon sources. Applied Biochemistry and Biotechnology, 161, 347–364.CrossRefGoogle Scholar
  25. 25.
    Su, X., Schmitz, G., Zhang, M., Mackie, R. I., & Cann, I. K. (2012). Heterologous gene expression in filamentous fungi. Advances in Applied Microbiology, 81, 1–61.CrossRefGoogle Scholar
  26. 26.
    Sumner, J. B. (1924). The estimation of sugar in diabetic urine using dinitrosalicylic acid. Journal of Biological Chemistry, 62(2), 287–290.Google Scholar
  27. 27.
    Teugjas, H., & Väljamäe, P. (2013). Selecting beta-glucosidases to support cellulases in cellulose saccharification. Biotechnology for Biofuels, 6, 105.CrossRefGoogle Scholar
  28. 28.
    Uchima, C. A., Tokuda, G., Watanabe, H., Kitamoto, K., & Arioka, M. (2011). Heterologous expression and characterization of a glucose-stimulated beta-glucosidase from the termite Neotermes koshunensis in Aspergillus oryzae. Applied Microbiology and Biotechnology, 89, 1761–1771.CrossRefGoogle Scholar
  29. 29.
    Uchima, C. A., Tokuda, G., Watanabe, H., Kitamoto, K., & Arioka, M. (2012). Heterologous expression in Pichia pastoris and characterization of an endogenous thermostable and high-glucose-tolerant beta-glucosidase from the termite Nasutitermes takasagoensis. Applied and Environment Microbiology, 78, 4288–4293.CrossRefGoogle Scholar
  30. 30.
    Wilson, D. B. (2009). Cellulases and biofuels. Current Opinion in Biotechnology, 20, 295–299.CrossRefGoogle Scholar
  31. 31.
    Znameroski, E. A., Coradetti, S. T., Roche, C. M., Tsai, J. C., Iavarone, A. T., Cate, J. H., et al. (2012). Induction of lignocellulose-degrading enzymes in Neurospora crassa by cellodextrins. Proceedings of the National Academy of Sciences of the United States of America, 109, 6012–6017.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Ana C. Colabardini
    • 1
    Email author
  • Mari Valkonen
    • 1
  • Anne Huuskonen
    • 1
  • Matti Siika-aho
    • 1
  • Anu Koivula
    • 1
  • Gustavo H. Goldman
    • 2
    • 3
  • Markku Saloheimo
    • 1
  1. 1.VTT Technical Research Centre of Finland LtdEspooFinland
  2. 2.Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão PrêtoUniversidade de São PauloRibeirão PrêtoBrazil
  3. 3.Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE)Centro Nacional de Pesquisa em Energia e Materiais (CNPEM)CampinasBrazil

Personalised recommendations