The Journal of Microbiology

, Volume 48, Issue 1, pp 53–62 | Cite as

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

  • Cesar Vanderlei Nascimento
  • Flávio Henrique Moreira Souza
  • Douglas Chodi Masui
  • Francisco Assis Leone
  • Rosane Marina Peralta
  • João Atílio Jorge
  • Rosa Prazeres Melo Furriel
Articles

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.

Keywords

cellobiase glucose-stimulated β-D-glucosidase H. grisea sugarcane bagasse agricultural residues thermophilic fungi 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 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.CrossRefGoogle Scholar
  2. Beguin, P. and J.P. Aubert. 1994. The biological degradation of cellulose. FEMS Microbiol. Rev. 13, 25–58.CrossRefPubMedGoogle Scholar
  3. 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
  4. Bhat, M. and T.S. Bhat. 1997. Cellulose degrading enzymes and their potential industrial applications. Biotechnol. Adv. 15, 583–620.CrossRefPubMedGoogle Scholar
  5. Bhatia, Y., S. Mishra, and V.S. Bisaria. 2002. Microbial β-glucosidases: cloning, properties, and applications. Crit. Rev. Biotechnol. 22, 375–407.CrossRefPubMedGoogle Scholar
  6. 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.CrossRefPubMedGoogle Scholar
  7. 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.Google Scholar
  8. Davis, B.J. 1964. Disc electrophoresis. II. Method and application to human serum proteins. Ann. N. Y. Acad. Sci. 121, 404–427.CrossRefPubMedGoogle Scholar
  9. 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.CrossRefPubMedGoogle Scholar
  10. 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.PubMedGoogle Scholar
  11. 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.CrossRefGoogle Scholar
  12. 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.CrossRefGoogle Scholar
  13. El-Hawary, F.I. and Y.S. Mostafa. 2001. Factors affecting cellulose production by Trichoderma koningii. Acta Aliment. 30, 3–13.CrossRefGoogle Scholar
  14. 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.CrossRefGoogle Scholar
  15. Galbe, M. and G. Zacchi. 2002. A review of the production of ethanol from softwood. Appl. Microbiol. Biotechnol. 59, 618–628.CrossRefPubMedGoogle Scholar
  16. 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.CrossRefGoogle Scholar
  17. 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.CrossRefPubMedGoogle Scholar
  18. Harchand, R.K. and S. Singh. 1997. Characterization of cellulose complex of Streptomyces albaduncus. J. Basic Microbiol. 37, 93–103.CrossRefPubMedGoogle Scholar
  19. 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.CrossRefPubMedGoogle Scholar
  20. Hayashida, S., K. Ohta, and K. Mo. 1988. Cellulases of Humicola insolens and Humicola grisea. Methods Enzymol. 160, 323–332.CrossRefGoogle Scholar
  21. 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.CrossRefGoogle Scholar
  22. 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.CrossRefPubMedGoogle Scholar
  23. 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.Google Scholar
  24. 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.Google Scholar
  25. 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.PubMedCrossRefGoogle Scholar
  26. 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.CrossRefPubMedGoogle Scholar
  27. Kumar, R., S. Singh, and O.V. Singh. 2008. Bioconversion of lingocellulosic biomass: biochemical and molecular perspectives. J. Ind. Microbiol. Biotechnol. 35, 377–391.CrossRefPubMedGoogle Scholar
  28. 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.CrossRefGoogle Scholar
  29. 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.CrossRefGoogle Scholar
  30. 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.CrossRefPubMedGoogle Scholar
  31. 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.PubMedGoogle Scholar
  32. 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
  33. 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.CrossRefPubMedGoogle Scholar
  34. Mandels, G.R. 1953. Localization of carbohydrases at the surface of fungus spores by acid treatment. Exp. Cell Res. 5, 48–55.CrossRefPubMedGoogle Scholar
  35. 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.CrossRefPubMedGoogle Scholar
  36. Masheshwari, R., G. Bharadwaj, and M.K. Bhat. 2000. Thermophilic fungi: their physiology and enzymes. Microbiol. Mol. Biol. Rev. 64, 461–488.CrossRefGoogle Scholar
  37. 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.CrossRefPubMedGoogle Scholar
  38. Miller, G.L. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugars. Anal. Chem. 31, 426–428.CrossRefGoogle Scholar
  39. 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.CrossRefPubMedGoogle Scholar
  40. 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.CrossRefGoogle Scholar
  41. 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
  42. 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.CrossRefPubMedGoogle Scholar
  43. 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.Google Scholar
  44. 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.PubMedGoogle Scholar
  45. 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.PubMedGoogle Scholar
  46. 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.CrossRefGoogle Scholar
  47. 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.PubMedGoogle Scholar
  48. 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.PubMedGoogle Scholar
  49. Saha, B.C. and R.J. Bothast. 1996a. Glucose tolerant and thermophilic β-glucosidases from yeasts. Biotechnol. Lett. 18, 155–158.CrossRefGoogle Scholar
  50. 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.PubMedGoogle Scholar
  51. 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.CrossRefPubMedGoogle Scholar
  52. 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.CrossRefGoogle Scholar
  53. 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.CrossRefPubMedGoogle Scholar
  54. 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.CrossRefPubMedGoogle Scholar
  55. Varki, A. 1993. Biological roles of oligosaccharides: all of the theories are correct. Glycobiology 3, 97–130.CrossRefPubMedGoogle Scholar
  56. 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.CrossRefPubMedGoogle Scholar
  57. Weber, K. and M. Osborn. 1969. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244, 4406–4412.PubMedGoogle Scholar
  58. 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.PubMedGoogle Scholar
  59. 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.CrossRefPubMedGoogle Scholar
  60. 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.CrossRefPubMedGoogle Scholar
  61. 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.CrossRefPubMedGoogle Scholar
  62. 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.CrossRefGoogle Scholar

Copyright information

© The Microbiological Society of Korea and Springer-Verlag Berlin Heidelberg 2010

Authors and Affiliations

  • Cesar Vanderlei Nascimento
    • 1
  • Flávio Henrique Moreira Souza
    • 1
  • Douglas Chodi Masui
    • 1
  • Francisco Assis Leone
    • 1
  • Rosane Marina Peralta
    • 2
  • João Atílio Jorge
    • 3
  • Rosa Prazeres Melo Furriel
    • 1
  1. 1.Department of Chemistry, Faculdade de Filosofia, Ciências e Letras de Ribeirão PretoUniversity of São PauloRibeirão PretoBrasil
  2. 2.Department of BiochemistryState University of MaringáMaringáBrasil
  3. 3.Department of Biology, Faculdade de Filosofia, Ciências e Letras de Ribeirão PretoUniversity of São PauloRibeirão PretoBrasil

Personalised recommendations