The Journal of Microbiology

, Volume 46, Issue 1, pp 51–55 | Cite as

Purification and characterization of thermostable β-glucosidase from the brown-rot basidiomycete Fomitopsis palustris grown on microcrystalline cellulose

Article

Abstract

An extracellular β-glucosidase was purified 154-fold to electrophoretic homogeneity from the brown-rot basidiomycete Fomitopsis palustris grown on 2.0% microcrystalline cellulose. SDS-polyacrylamide gel electrophoresis gel gave a single protein band and the molecular mass of purified enzyme was estimated to be approximately 138 kDa. The amino acid sequences of the proteolytic fragments determined by nano-LC-MS/MS suggested that the protein has high homology with fungal β-glucosidases that belong to glycosyl hydrolase family 3. The Kms for p-nitorophenyl-β-d-glucoside (p-NPG) and cellobiose hydrolyses were 0.117 and 4.81 mM, and the Kcat values were 721 and 101.8 per sec, respectively. The enzyme was competitively inhibited by both glucose (Ki= 0.35 mM) and gluconolactone (Ki 0.008 mM), when p-NPG was used as substrate. The optimal activity of the purified β-glucosidase was observed at pH 4.5 and 70°C. The F. palustris protein exhibited half-lives of 97 h at 55°C and 15 h at 65°C, indicating some degree of thermostability. The enzyme has high activity against p-NPG and cellobiose but has very little or no activity against p-nitrophenyl-β-lactoside, p-nitrophenyl-β-xyloside, p-nitrophenyl-α-arabinofuranoside, xylan, and carboxymethyl cellulose. Thus, our results revealed that the β-glucosidase from F. palustris can be classified as an aryl-β-glucosidase with cellobiase activity.

Keywords

β-glucosidase brown-rot fungus Fomitopsis palustris purification microcrystalline cellulose 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Abe, S. and M. Takagi. 1991. Simultaneous saccharification and fermentation of cellulose to lactic acid. Biotechnol. Bioeng. 37, 93–96.CrossRefPubMedGoogle Scholar
  2. 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.PubMedCrossRefGoogle Scholar
  3. Cai, Y.J., J.A. Buswell, and S.T. Chang. 1998. β-Glucosidase components of the cellulolytic system of the edible straw mushroom, Volvariella volvacea. Enzyme Microb. Technol. 22, 122–129.CrossRefGoogle Scholar
  4. Cantarella, M., L. Cantarella, A. Gallifuoco, A. Spera, and F. Alfani. 2004. Effect of inhibitors released during steam-explosion treatment of poplar wood on subsequent enzymatic hydrolysis and SSF. Biotechnol. Prog. 20, 200–206.PubMedCrossRefGoogle Scholar
  5. Chen, H., M. Hayn, and H. Esterbauer. 1992. Purification and characterization of two extracellular beta-glucosidases from Trichoderma reesei. Biochim. Biophys. Acta. 1121, 54–60.PubMedGoogle Scholar
  6. Chirico, W.J. and R.D. Brown, Jr. 1987. Purification and characterization of a beta-glucosidase from Trichoderma reesei. Eur. J. Biochem. 165, 343–351.PubMedCrossRefGoogle Scholar
  7. Claeyssens, M., H. Van Tilbeurgh, P. Tomme, T.M. Wood, and I. McCrae. 1989. Fungal cellulase systems. Comparison of the specificities of the cellobiohydrolases isolated from Penicillium pinophilum and Trichoderma reesei. Biochem. J. 261, 819–826.PubMedGoogle Scholar
  8. Eriksson, K.-E.L., R.A. Blanchette, and P. Ander. 1990. Microbial and enzymatic degradation of wood and wood components. Springer Verlag, Berlin/Heidelberg, Germany.Google Scholar
  9. Lee, J.-W., J.-Y. Park, K.-S. Gwak, B.-W. Koo, and I.-G. Choi. 2007. Characterization of β-glucosidase from brown rot fungus, Laetiporus sulphureus. Korean J. Wood Sci. Technol. 35, 100–108.Google Scholar
  10. Lo, A.C., G. Willick, R. Bernier, and M. Desrochers. 1988. Purification and assay of β-glucosidase from Schizophyllum commune. Methods Enzymol. 160, 432–437.CrossRefGoogle Scholar
  11. Lymar, E.S., B. Li, and V. Renganathan. 1995. Purification and characterization of a cellulose-binding (beta)-glucosidase from cellulose-degrading cultures of Phanerochaete chrysosporium. Appl. Environ. Microbiol. 61, 2976–2980.PubMedGoogle Scholar
  12. Magalhães, P.O., A. Ferraz, and A.F.M. Milaqres. 2006. Enzymatic properties of two beta-glucosidases from Ceriporiopsis subvermispora produced in biopulping conditions. J. Appl. Microbiol. 101, 480–486.PubMedCrossRefGoogle Scholar
  13. Martinez, G., L.F. Larrondo, N. Putnam, M.D.S. Gelpke, K. Huang, J. Chapman, K.G. Helfenbein, P. Ramaiya, J.C. Detter, F. Larimer, P.M. Coutinho, B. Henrissat, R. Berka, D. Cullen, and D. Rokhsar. 2004. Genome sequence of the lignocellulose degrading fungus Phanerochaete chrysosporium strain RP78. Nature Biotechnol. 22, 1–6.CrossRefGoogle Scholar
  14. Saha, B.C., S.N. Freer, and R.J. Bothast. 1994. Production, purification, and properties of a thermostable beta-glucosidase from a color variant strain of Aureobasidium pullulans. Appl. Environ. Microbiol. 60, 3774–3780.PubMedGoogle Scholar
  15. Shevchenko, A., M. Wilm, O. Vorm, and M. Mann. 1996. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68, 850–858.PubMedCrossRefGoogle Scholar
  16. Uzcategui, E., G. Johansson, B. Ek, and G. Pettersson. 1991. The 1,4-β-d-glucan glucanohydrolases from Phanerochaete chrysosporium. Reassessment of their significance in cellulose degradation mechanisms. J. Biotechnol. 21, 143–159.PubMedCrossRefGoogle Scholar
  17. Woodward, J., M. Lima, and N.E. Lee. 1982. The role of cellulose concentration in determining the degree of synergism in the hydrolysis of microcrystalline cellulose. Biochem. J. 255, 895–899.Google Scholar
  18. Yoon, J.-J., C.-J. Cha, Y.-S. Kim, D.-W. Son, and Y.-K. Kim. 2007. The brown-rot basidiomycete Fomitopsis palustris has the endo-glucanases capable of degrading microcrystalline cellulose. J. Microbiol. Biotechnol. 17, 800–805.PubMedGoogle Scholar
  19. Yoon, J.-J. and Y.-K. Kim. 2005. Degradation of crystalline cellulose by the brown-rot basidiomycete Fomitopsis palustris. J. Microbiol. 43, 487–492.PubMedGoogle Scholar

Copyright information

© The Microbiological Society of Korea 2008

Authors and Affiliations

  1. 1.Department of Bioscience and BiotechnologyKonkuk UniversitySeoulRepublic of Korea
  2. 2.Department of Biotechnology and BET InstituteChung-Ang UniversityAnseongRepublic of Korea

Personalised recommendations