Skip to main content
SpringerLink
Log in

Purification and biochemical properties of a glucose-stimulated β-D-glucosidase produced by Humicola grisea var. thermoidea grown on sugarcane bagasse

  • Articles
  • Published:
The Journal of Microbiology Aims and scope Submit manuscript

Abstract

The effect of several carbon sources on the production of mycelial-bound β-glucosidase by Humicola grisea var. thermoidea in submerged fermentation was investigated. Maximum production occurred when cellulose was present in the culture medium, but higher specific activities were achieved with cellobiose or sugarcane bagasse. Xylose or glucose (1%) in the reaction medium stimulated β-glucosidase activity by about 2-fold in crude extracts from mycelia grown in sugarcane bagasse. The enzyme was purified by ammonium sulfate precipitation, followed by Sephadex G-200 and DEAE-cellulose chromatography, showing a single band in PAGE and SDS-PAGE. The β-glucosidase had a carbohydrate content of 43% and showed apparent molecular masses of 57 and 60 kDa, as estimated by SDS-PAGE and gel filtration, respectively. The optimal pH and temperature were 6.0 and 50°C, respectively. The purified enzyme was thermostable up to 60 min in water at 55°C and showed half-lives of 7 and 14 min when incubated in the absence or presence of 50 mM glucose, respectively, at 60°C. The enzyme hydrolyzed p-nitrophenyl-β-D-glucopyranoside, p-nitrophenyl-β-Dgalactopyranoside, p-nitrophenyl-β-D-fucopyranoside, p-nitrophenyl-β-D-xylopyranoside, o-nitrophenyl-β-Dgalactopyranoside, lactose, and cellobiose. The best synthetic and natural substrates were p-nitrophenyl-β-Dfucopyranoside and cellobiose, respectively. Purified enzyme activity was stimulated up to 2-fold by glucose or xylose at concentrations from 25 to 200 mM. The addition of purified or crude β-glucosidase to a reaction medium containing Trichoderma reesei cellulases increased the saccharification of sugarcane bagasse by about 50%. These findings suggest that H. grisea var. thermoidea β-glucosidase has a potential for biotechnological applications in the bioconversion of lignocellulosic materials.

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

Similar content being viewed by others

References

  • Andrade, S.V., M.L.T.M. Polizeli, H.F. Terenzi, and J.A. Jorge. 2004. Effect of carbon source on the biochemical properties of β-xylosidases produced by Aspergillus versicolor. Proc. Biochem. 39, 1931–1938.

    Article  CAS  Google Scholar 

  • Beguin, P. and J.P. Aubert. 1994. The biological degradation of cellulose. FEMS Microbiol. Rev. 13, 25–58.

    Article  PubMed  CAS  Google Scholar 

  • Bergmeyer, H.U. and E. Bernt. 1974. D-glucose determination with glucose oxidase and peroxidase, pp. 1205–1215. In H.U. Bergmeyer (ed.), Methods of Enzymatic Analysis, vol. 3. Verlag Chimie-Academic Press, New York, N.Y., USA.

    Google Scholar 

  • Bhat, M. and T.S. Bhat. 1997. Cellulose degrading enzymes and their potential industrial applications. Biotechnol. Adv. 15, 583–620.

    Article  PubMed  CAS  Google Scholar 

  • Bhatia, Y., S. Mishra, and V.S. Bisaria. 2002. Microbial β-glucosidases: cloning, properties, and applications. Crit. Rev. Biotechnol. 22, 375–407.

    Article  PubMed  CAS  Google Scholar 

  • Bhiri, F., S.E. Chaabouni, F. Limam, R. Ghrir, and N. Marzouki. 2008. Purification and biochemical characterization of extracellular β-glucosidases from the hypercellulolytic Pol6 mutant of Penicillium occitanis. Appl. Biochem. Biotechnol. 149, 169–182.

    Article  PubMed  CAS  Google Scholar 

  • Cooney, D.G. and R. Emerson. 1964. Humicola insolens and Humicola grisea var. thermoidea. Thermophilic fungi: an account of their biology, activities, and classification, pp. 73–79. In W.H. Freeman (ed.), San Francisco, California, USA.

  • Davis, B.J. 1964. Disc electrophoresis. II. Method and application to human serum proteins. Ann. N. Y. Acad. Sci. 121, 404–427.

    Article  PubMed  CAS  Google Scholar 

  • Decker, C.H., J. Visser, and P. Schreier. 2001. β-glucosidase multiplicity from Aspergillus tubingiensis CBS 643.92: purification and characterization of four β-glucosidases and their differentiation with respect to substrate specificity, glucose inhibition and acid tolerance. Appl. Microbiol. Biotechnol. 55, 157–163.

    Article  PubMed  CAS  Google Scholar 

  • Dey, N.B., P. Bounelis, T.A. Fritz, D.M. Bedewell, and R.B. Marchase. 1994. The glycosylation of phosphoglumutase is modulated by carbon source and heat shock in Saccharomyces cerevisiae. J. Biol. Chem. 269, 27143–27148.

    PubMed  CAS  Google Scholar 

  • Dubois, M., K.A. Gilles, J.K. Hamilton, P.A. Rebers, and F. Smith. 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350–356.

    Article  CAS  Google Scholar 

  • Duff, S.J.B. and W.D. Murray. 1996. Bioconversion of forest products industry waste cellulosics to fuel ethanol: A review. Biores. Technol. 55, 1–33.

    Article  CAS  Google Scholar 

  • El-Hawary, F.I. and Y.S. Mostafa. 2001. Factors affecting cellulose production by Trichoderma koningii. Acta Aliment. 30, 3–13.

    Article  CAS  Google Scholar 

  • El-Hawary, F.I., Y.S. Mostafa, and E. Laszlo. 2001. Cellulase production and conversion of rice straw to lactic acid by simultaneous saccharification and fermentation. Acta Aliment. 30, 281–295.

    Article  CAS  Google Scholar 

  • Galbe, M. and G. Zacchi. 2002. A review of the production of ethanol from softwood. Appl. Microbiol. Biotechnol. 59, 618–628.

    Article  PubMed  CAS  Google Scholar 

  • Gao, J., H. Weng, D. Zhu, M. Yuan, F. Guan, and Y. Xi. 2008. Production and characterization of cellulolytic enzymes from the thermoacidophilic fungal Aspergillus terreus M11 under solid-state cultivation on corn stover. Biores. Technol. 99, 7623–7629.

    Article  CAS  Google Scholar 

  • Gusakov, A.V., T.N. Salanovich, A.I. Antonov, B.B. Ustinov, O.N. Okunev, R. Burlingame, M. Emalfarb, M. Baez, and A.P. Sinitsyn. 2007. Design of highly efficient cellulase mixtures for enzymatic hydrolysis of cellulose. Biotechnol. Bioeng. 97, 1028–1038.

    Article  PubMed  CAS  Google Scholar 

  • Harchand, R.K. and S. Singh. 1997. Characterization of cellulose complex of Streptomyces albaduncus. J. Basic Microbiol. 37, 93–103.

    Article  PubMed  CAS  Google Scholar 

  • Harrison, M.J., A.S. Nouwens, D.R. Jardine, N.E. Zachara, A.A. Gooley, and H. Nevalainen. 1998. Modified glycosylation of cellobiohydrolase I from a high cellulase producing mutant strain of Trichoderma reesei. Eur. J. Biochem. 256, 119–127.

    Article  PubMed  CAS  Google Scholar 

  • Hayashida, S., K. Ohta, and K. Mo. 1988. Cellulases of Humicola insolens and Humicola grisea. Methods Enzymol. 160, 323–332.

    Article  CAS  Google Scholar 

  • Hui, J.P.M., P. Lanthier, T.C. White, S.G. McHugh, M. Yaguchi, R. Roy, and P. Tribault. 2001. Characterization of cellobiohydrolase I (Cel7A) glycoforms from extracts of Trichoderma reesei using capillary isoelectric focusing and electrospray mass spectrometry. J. Chrom. B 752, 349–368.

    Article  CAS  Google Scholar 

  • Karnchanatat, A., A. Petsom, P. Sangvanich, J. Piaphukiew, A.J. Whalley, C.D. Reynolds, and P. Sihanonth. 2007. Purification and biochemical characterization of an extracellular beta-glucosidase from the wood-decaying fungus Daldinia eschscholzii (Ehrenb.: Fr.) Rehm. FEMS Microbiol. Lett. 270, 162–170.

    Article  PubMed  CAS  Google Scholar 

  • Kaur, J., B.S. Chadha, B.A. Kumar, S.K. Ghatora, and H.S. Saini. 2007. Purification and characterization of β-glucosidase from Melanocarpus sp. MTCC 3922. Electronic J. Biotechnol. 10, 260–270.

    CAS  Google Scholar 

  • Kaur, J., B.S. Chadha, and H.S. Saini. 2006. Regulation of cellulose production in two thermophilic fungi Melanocarpus sp. MTCC 3922 and Scytalidium thermophilum MTCC 4520. Enzyme Microb. Technol. 38, 931–936.

    CAS  Google Scholar 

  • Kern, G., N. Schülke, F.X. Schmid, and R. Jaenicke. 1992. Stability, quaternary structure, and folding of internal, external, and coreglycosylated invertase from yeast. Protein Sci. 1, 120–131.

    Article  PubMed  CAS  Google Scholar 

  • Klarskov, K., K. Piens, J. Stahlberg, P.B. Hoj, J.M. Van Beeumen, and M. Claeyssens. 1997. Cellobiohydrolase I from Trichoderma reesei: Identification of an active-site nucelophile and additional information on sequence including the glycosylation pattern for the core protein. Carbohydr. Res. 304, 143–154.

    Article  PubMed  CAS  Google Scholar 

  • Kumar, R., S. Singh, and O.V. Singh. 2008. Bioconversion of lingocellulosic biomass: biochemical and molecular perspectives. J. Ind. Microbiol. Biotechnol. 35, 377–391.

    Article  PubMed  CAS  Google Scholar 

  • Leite, R.S.R., H.F. Alves-Prado, H. Cabral, F.C. Pagnocca, E. Gomes, and R. Da-Silva. 2008. Production and characteristics comparison of crude β-glucosidases produced by microorganisms Thermoascus aurantiacus e Aureobasidium pullulans in agricultural wastes. Enzyme Microb. Technol. 43, 391–395.

    Article  CAS  Google Scholar 

  • Leone, F.A., J.A. Baranauskas, R.P.M. Furriel, and I.A. Borin. 2005. SigrafW: an easy-to-use program for fitting enzyme kinetic data. Biochem. Mol. Biol. Educ. 33, 399–403.

    Article  CAS  Google Scholar 

  • Lige, B., S. Ma, and R.B. van Huystee. 2001. The effects of the sitedirected removal of N-glycosylation from cationic peanut peroxidase on its function. Arch. Biochem. Biophys. 386, 17–24.

    Article  PubMed  CAS  Google Scholar 

  • Lin, J., B. Pillay, and S. Singh. 1999. Purification and biochemical characteristics of β-D-glucosidase from a thermophilic fungus Thermomyces lanuginosus-SSBP. Biotechnol. Appl. Biochem. 30, 81–87.

    PubMed  CAS  Google Scholar 

  • Lowry, O.H., N.J. Rosebrough, A.L. Farr, and R.J. Randall. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 13, 265–275.

    Google Scholar 

  • Lynd, L.R., P.J. Weimer, W.H. Zyl, and I.S. Pretorius. 2002. Microbial cellulose utilization: fundamentals and biotechnology. Microbiol. Mol. Biol. Rev. 66, 506–577.

    Article  PubMed  CAS  Google Scholar 

  • Mandels, G.R. 1953. Localization of carbohydrases at the surface of fungus spores by acid treatment. Exp. Cell Res. 5, 48–55.

    Article  PubMed  CAS  Google Scholar 

  • Maras, M., A. DeBruyn, J. Schraml, P. Herdewijn, M. Claeyssens, W. Fiers, and R. Contreras. 1997. Structural characterization of Nlinked oligosaccharides from cellobiohydrolase secreted by filamentous fungi Trichoderma reesei Rut-C-30. Eur. J. Biochem. 245, 617–625.

    Article  PubMed  CAS  Google Scholar 

  • Masheshwari, R., G. Bharadwaj, and M.K. Bhat. 2000. Thermophilic fungi: their physiology and enzymes. Microbiol. Mol. Biol. Rev. 64, 461–488.

    Article  Google Scholar 

  • Meldgaard, M. and I. Svendsen. 1994. Different effects of Nglycosylation on the thermostability of highly homologous bacterial (1,3-1,4)-β-glucananases secreted from yeast. Microbiology 140, 159–166.

    Article  PubMed  CAS  Google Scholar 

  • Miller, G.L. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugars. Anal. Chem. 31, 426–428.

    Article  CAS  Google Scholar 

  • Nakkharat, P. and D. Haltrich. 2006. Purification and characterization of an intracellular enzyme with β-glucosidase and β-galactosidase activity from the thermophilic fungus Talaromyces thermophilus CBS 236.58. J. Biotechnol. 123, 304–313.

    Article  PubMed  CAS  Google Scholar 

  • Nieves, R.A., C.I. Ehrman, W.S. Adney, R.T. Elander, and M.E. Himmel. 1998. Technical communication: Survey and analysis of commercial cellulose preparations suitable for biomass conversion to ethanol. World J. Microbiol. Biotechnol. 14, 301–304.

    Article  CAS  Google Scholar 

  • Osaki, H. and K. Yamada. 1991. Isolation of Streptomyces sp. producing glucose-tolerant β-glucosidases and properties of the enzymes. Agric. Biol. Chem. 55, 979–987.

    Google Scholar 

  • Parry, N.J., D.E. Beever, E. Owen, I. Vandenberghe, J. Van Beeumen, and M.K. Bhat. 2001. Biochemical characterization and mechanisms of action of a thermostable β-glucosidase purified from Thermoascus aurantiacus. Biochem. J. 353, 117–127.

    Article  PubMed  CAS  Google Scholar 

  • Peralta, R.M., M.K. Kadowaki, H.F. Terenzi, and J.A. Jorge. 1997. A highly thermostable β-glucosidase activity from the thermophilic fungus Humicola grisea var. thermoidea: purification and biochemical characterization. FEMS Microbiol. Lett. 146, 291–295.

    CAS  Google Scholar 

  • Peralta, R.M., H.F. Terenzi, and J.A. Jorge. 1990. β-Glycosidase activities of Humicola grisea: biochemical and kinetic characterization of a multifunctional enzyme. Biochim. Biophys. Acta. 1033, 243–249.

    PubMed  CAS  Google Scholar 

  • Perez-Pons, J.A., X. Rebordosa, and E. Querol. 1995. Properties of a novel glucose-enhanced β-glucosidase purified from Streptomyces sp. (ATCC 11238). Biochim. Biophys. Acta. 1251, 145–153.

    PubMed  Google Scholar 

  • Polizeli, M.L.T.M., J.A. Jorge, and H.F. Terenzi. 1996. Effect of carbon source on the β-glucosidase system of the thermophilic fungus Humicola grisea. World J. Microbiol. Biotechnol. 12, 297–299.

    Article  CAS  Google Scholar 

  • Rao, U.S. and S.K. Murthy. 1988. Purification and characterization of a beta-glucosidase and endocellulase from Humicola insolens. Indian J. Biochem. Biophys. 25, 687–694.

    PubMed  CAS  Google Scholar 

  • Riou, C., J.M. Salmon, M.J. Vallier, Z. Günata, and P. Bare. 1998. Purification, characterization and substrate specificity of a novel highly glucose-tolerant β-glucosidase from Aspergillus oryzae. Appl. Environ. Microbiol. 64, 3607–3614.

    PubMed  CAS  Google Scholar 

  • Saha, B.C. and R.J. Bothast. 1996a. Glucose tolerant and thermophilic β-glucosidases from yeasts. Biotechnol. Lett. 18, 155–158.

    Article  CAS  Google Scholar 

  • Saha, B.C. and R.J. Bothast. 1996b. Production, purification and characterization of a highly glucose-tolerant novel β-glucosidase from Candida peltata. Appl. Environ. Microbiol. 62, 3165–3170.

    PubMed  CAS  Google Scholar 

  • Somera, A.F., M.G. Pereira, L.H.S. Guimarães, M.L.T.M. Polizeli, H.F. Terenzi, R.P.M. Furriel RPM, and J.A. Jorge. 2009. Effect of glycosylation on the biochemical properties of Aspergillus versicolor. J. Microbiol. 47, 270–276.

    Article  PubMed  CAS  Google Scholar 

  • Sonia, K.G., B.S. Chadha, A.K. Badhan, H.S. Saini, and M.K. Bhat. 2008. Identification of glucose tolerant acid active β-glucosidases from thermophilic and thermotolerant fungi. World J. Microbiol. Biotechnol. 24, 599–604.

    Article  CAS  Google Scholar 

  • Stals, I., K. Sandra, B. Devreese, J. Van Beeumen, and M. Claeyssens. 2004a. Factors influencing glycosylation of Trichoderma reesei cellulases. II: N-glycosylation of Cel7A core protein isolated from different strains. Glycobiology 14, 725–737.

    Article  PubMed  CAS  Google Scholar 

  • Stals, I., K. Sandra, S. Geysens, R. Contreras, J. Van Beeumen, and M. Claeyssens. 2004b. Factors influencing glycosylation of Trichoderma reesei cellulases. I: Postsecretorial changes of the O- and N-glycosylation pattern of Cel7A. Glycobiology 14, 713–724.

    Article  PubMed  CAS  Google Scholar 

  • Varki, A. 1993. Biological roles of oligosaccharides: all of the theories are correct. Glycobiology 3, 97–130.

    Article  PubMed  CAS  Google Scholar 

  • Venturi, L.L., M.L.T.M. Polizeli, H.F. Terenzi, R.P.M. Furriel, and J.A. Jorge. 2002. Extracellular β-D-glucosidase from Chaetomium thermophilum var. coprophilum: production, purification and some biochemical properties. J. Basic Microbiol. 42, 55–66.

    Article  PubMed  CAS  Google Scholar 

  • Weber, K. and M. Osborn. 1969. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244, 4406–4412.

    PubMed  CAS  Google Scholar 

  • Wright, R.M., M.D. Yablonsky, Z.P. Shalita, A.K. Goyal, and D.E. Eveleigh. 1992. Cloning, characterization, nucleotide sequence of a gene encoding Microbispora bispora BglB, a thermostable β-glucosidase expressed in Escherichia coli. Appl. Environ. Microbiol. 58, 3455–3465.

    PubMed  CAS  Google Scholar 

  • Yang, S., Z. Jiang, Q. Yan, and H. Zhu. 2008. Characterization of a thermostable extracellular β-glucosidase with activities of exoglucanase and transglycosylation from Paecilomyces thermophila. J. Agric. Food Chem. 56, 602–608.

    Article  PubMed  CAS  Google Scholar 

  • Yoon, J.J., K.Y. Kim, and C.J. Cha. 2008. Purification and characterization of thermostable β-glucosidase from the brown-rot basidiomycete Fomitopsis palustris grown on microcrystalline cellulose. J. Microbiol. 46, 51–55.

    Article  PubMed  CAS  Google Scholar 

  • Zanoelo, F.F., M.L.T.M. Polizeli, H.F. Terenzi, and J.A. Jorge. 2004. β-Glucosidase activity from the thermophilic fungus Scytalidium thermophilum is stimulated by glucose and xylose. FEMS Microbiol. Lett. 240, 137–143.

    Article  PubMed  CAS  Google Scholar 

  • Zhang, Y.H.P., M.E. Himmel, and J.R. Mielenz. 2006. Outlook for cellulase improvement: screening and selection strategies. Biotechnol. Adv. 24, 452–481.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Chemistry, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, University of São Paulo, Ribeirão Preto, 14040-901, SP, Brasil

    Cesar Vanderlei Nascimento, Flávio Henrique Moreira Souza, Douglas Chodi Masui, Francisco Assis Leone & Rosa Prazeres Melo Furriel

  2. Department of Biochemistry, State University of Maringá, 87020-900, Maringá, PR, Brasil

    Rosane Marina Peralta

  3. Department of Biology, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, University of São Paulo, Ribeirão Preto, 14040-901, SP, Brasil

    João Atílio Jorge

Authors
  1. Cesar Vanderlei Nascimento
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Flávio Henrique Moreira Souza
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Douglas Chodi Masui
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Francisco Assis Leone
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Rosane Marina Peralta
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. João Atílio Jorge
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Rosa Prazeres Melo Furriel
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Rosa Prazeres Melo Furriel.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Nascimento, C.V., Souza, F.H.M., Masui, D.C. et al. Purification and biochemical properties of a glucose-stimulated β-D-glucosidase produced by Humicola grisea var. thermoidea grown on sugarcane bagasse. J Microbiol. 48, 53–62 (2010). https://doi.org/10.1007/s12275-009-0159-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12275-009-0159-x

Keywords

Search

Navigation