Purification and biochemical properties of a thermostable xylose-tolerant β-D-xylosidase from Scytalidium thermophilum

  • Fabiana Fonseca Zanoelo
  • Maria de Lourdes Teixeira de Moraes Polizeli
  • Héctor Francisco Terenzi
  • João Atílio Jorge
Original Paper

Abstract

The thermophilic fungus Scytalidium thermophilum produced large amounts of periplasmic β-D-xylosidase activity when grown on xylan as carbon source. The presence of glucose in the fresh culture medium drastically reduced the level of β-D-xylosidase activity, while cycloheximide prevented induction of the enzyme by xylan. The mycelial β-xylosidase induced by xylan was purified using a procedure that included heating at 50°C, ammonium sulfate fractioning (30–75%), and chromatography on Sephadex G-100 and DEAE-Sephadex A-50. The purified β-D-xylosidase is a monomer with an estimated molecular mass of 45 kDa (SDS-PAGE) or 38 kDa (gel filtration). The enzyme is a neutral protein (pI 7.1), with a carbohydrate content of 12% and optima of temperature and pH of 60°C and 5.0, respectively. β-D-Xylosidase activity is strongly stimulated and protected against heat inactivation by calcium ions. In the absence of substrate, the enzyme is stable for 1 h at 60°C and has half-lives of 11 and 30 min at 65°C in the absence or presence of calcium, respectively. The purified β-D-xylosidase hydrolyzed p-nitrophenol-β-D-xylopyranoside and p-nitrophenol-β-D-glucopyranoside, exhibiting apparent Km and Vmax values of 1.3 mM, 88 μmol min−1 protein−1 and 0.5 mM, 20 μmol min−1 protein−1, respectively. The purified enzyme hydrolyzed xylobiose, xylotriose, and xylotetraose, and is therefore a true β-D-xylosidase. Enzyme activity was completely insensitive to xylose, which inhibits most β-xylosidases, at concentrations up to 200 mM. Its thermal stability and high xylose tolerance qualify this enzyme for industrial applications. The high tolerance of S. thermophilum β-xylosidase to xylose inhibition is a positive characteristic that distinguishes this enzyme from all others described in the literature.

Keywords

β-D-Xylosidase Xylanolytic activity Scytalidium thermophilum Thermostability 

References

  1. 1.
    Almeida EM, Polizeli MLTM, Terenzi HF, Jorge JA (1995) Purification and biochemical characterization of β-xylosidase from Humicola grisea var. thermoidea. FEMS Microbiol Lett 130:171–176CrossRefGoogle Scholar
  2. 2.
    Aquino ACMM, Jorge JA, Terenzi HF, Polizeli MLTM (2001) Thermostable glucose-tolerant glucoamylase produced by the thermophilic fungus Scytalidium thermophilum. Folia Microbiol (Praha) 46:11–16Google Scholar
  3. 3.
    Aquino ACMM, Jorge JA, Terenzi HF, Polizeli MLTM (2003) Studies on a thermostable α-amylase from the thermophilic fungus Scytalidium thermophilum. Appl Microbiol Biotechnol 61:323–328PubMedGoogle Scholar
  4. 4.
    Bütner R, Bode R (1992) Purification and characterization of β-xylosidase activities from the yeast Arxula adeninivorans. J Basic Microbiol 32:159–166PubMedGoogle Scholar
  5. 5.
    Cai YJ, Buswell JA, Chang ST (1998) β-Glucosidase components of the cellulolytic system of the edible straw mushroom, Volvariella volvacea. Enzyme Microb Technol 22:122–129CrossRefGoogle Scholar
  6. 6.
    Davis BJ (1964) Disc electropohoresis. II. Method and application to human serum proteins. Ann N Y Acad Sci 121:4040–427Google Scholar
  7. 7.
    Deshpande V, Lachke A, Misha C, Keskar S, Rao M (1986) Mode of action ande properties of xylanase and β-xylosidase from Neurospora crassa. Biotechnol Bioeng 28:1832–1837Google Scholar
  8. 8.
    Dobberstein J, Emeis CC (1991) Purification and characterization of β-xylosidase from Aureobasidium pullulans. Appl Microbiol Biotechnol 35:210–215Google Scholar
  9. 9.
    Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal chem 28:350–356Google Scholar
  10. 10.
    Ghosh M, Das A, Mishra AK, Nanda G (1993) Aspergillus sydowii MG 49 is strong producer of thermostable xylanolytic enzymes. Enzyme Microbiol Technol 15:703–709CrossRefGoogle Scholar
  11. 11.
    Guimarães LHS, Terenzi HF, Jorge JA, Polizeli MLTM (2001) Thermostable conidial and mycelial alkaline phosphatase from the thermophilic fungus Scytalidium thermophilum. J Ind Microbiol Biotechnol 27:265–270CrossRefPubMedGoogle Scholar
  12. 12.
    Hebraud M, Fevre M (1990) Purification and characterization of an extracellular β-xylosidase from the rumen anaerobic fungus Neocallimastix frontalis. FEMS Microbiol Lett 72:11–16CrossRefGoogle Scholar
  13. 13.
    Hermann MC, Vrsanska M, Jurickova M, Hirsch J, Biely P, Kubicek CP (1997) The β-D-xylosidase of Trichoderma reesei is a multifunctional β-D-xylan xylohydrolase. Biochem J 321:375–381PubMedGoogle Scholar
  14. 14.
    Iwashita K, Todoroki K, Kimura H, Shimoi H, Ito K (1998) Purification and characterization of extracellular and cell wall bound β-glucosidases from Aspergillus kawachii. Biosc Biotechnol Biochem 62:1938–1946Google Scholar
  15. 15.
    John M, Schmidt B, Schmidt J (1979) Purification and some properties of five endo-1,4-β-xylanases and a β-D-xylosidase produced by a strain of Aspergillus niger. Can J Biochem 57:125–134PubMedGoogle Scholar
  16. 16.
    Kadowaki MK, Polizeli MLTM, Terenzi HF, Jorge JA (1996) Characterization of trehalase activities from the thermophilic fungus Scytalidium thermophilum. Biochim Biophys Acta 1291:199–205CrossRefPubMedGoogle Scholar
  17. 17.
    Kimura I, Yoshioka N, Tajima S (1999) Purification and characterization of a β-glucosidase with β-xylosidase activity from Aspergillus sojae. J Biosci Bioeng 87:538–541CrossRefGoogle Scholar
  18. 18.
    Kulkarni N, Shendye A, Rao M (1999) Molecular and biotechnological aspects of xylanases. FEMS Microbiol Rev 23:411–456PubMedGoogle Scholar
  19. 19.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bateriophage T4. Nature 227:680–685PubMedGoogle Scholar
  20. 20.
    Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with folin phenol reagent. J Biol Chem 193:265–275Google Scholar
  21. 21.
    Maheshwari R, Bharadwaj G, Bhat MK (2000) Thermophilic fungi: their physiology and enzymes. Microbiol Mol Biol Rev 64:461–488PubMedGoogle Scholar
  22. 22.
    Mandels GR (1953) Localization of carbohydrases at the surface of fungus spores by acid treatment. Exp Cell Res 5:48–55PubMedGoogle Scholar
  23. 23.
    McIlvaine TC (1921) A buffer solution for colorimetric comparison. J Biol Chem 49:183–186Google Scholar
  24. 24.
    Monti R, Terenzi HF, Jorge JA (1991) Purification and properties of an extracellular xylanase from the thermophilic fungus Humicola grisea var. thermoidea. Can J Microbiol 37:675–681Google Scholar
  25. 25.
    Öscan S, Kötter P, Ciriacy M (1991) Xylan-hydrolysing enzymes of the yeast Pichia stipitis. Appl Microbiol Biotechnol 36:190–195Google Scholar
  26. 26.
    Peralta RM, Kadowaki MK, Terenzi HF, Jorge JA (1997) A highly thermostable β-glucosidase activity from thermophilic fungus Humicola grisea var. thermoidea: purification and biochemical characterization. FEMS Microbiol lett 146:291–295CrossRefGoogle Scholar
  27. 27.
    Poutanen K, Pulls J (1988) Characteristics of Trichoderma reesei β-xylosidase and its use in hydrolysis of solubilized xylans. Appl Microbiol Biotechnol 28:425–432Google Scholar
  28. 28.
    Poutanen K, Ratto M, Pulls P, Viikari L (1987) Evalualtion of different microbial xylanolytic system. J Biotechnol 6:49–60Google Scholar
  29. 29.
    Rizzati ACS, Jorge JA, Terenzi HF, Rechia CGV, Polizeli, MLTM (2001) Purification and properties of a thermostable extracellular β-xylosidase produced by a thermotolerant Aspergillus phoenicis. J Ind Microbiol Biotechnol 26:1–5CrossRefGoogle Scholar
  30. 30.
    Rosemberg SL (1978) Cellulose and lignocellulose degradation by thermophilic and thermotolerant fungi. Mycologia 70:1–13Google Scholar
  31. 31.
    Saha BC (2003) Hemicellulose bioconversion. J Ind Microbiol Biotechnol 30:279–291CrossRefPubMedGoogle Scholar
  32. 32.
    Straatsma G, Samson RA (1993) Taxonomy of Scytalidium thermophilum, an important thermophilic fungus in mushroom compost. Mycol Res 97:321–328Google Scholar
  33. 33.
    Sunna A, Antranikian G (1997) Xylanolytic enzymes from fungi and bacteria. Crit Rev Biotechnol 17:39–67PubMedGoogle Scholar
  34. 34.
    Van Peij NNME, Brinkman J, Vrsanská M, Visser J, de Graaff LH (1997) β-xylosidase activity, encoded by xlnD, is essential for complete hydrolysis of xylan by Aspergillus niger but not for induction of the xylanolytic enzyme spectrum. Eur J Biochem 245:164–173PubMedGoogle Scholar
  35. 35.
    Viikari L, Kantelinen A, Sundquist J, Linko M (1994) Xylanases in bleaching from a idea to the industry. FEMS Microbiol Rev 13:335–350CrossRefGoogle Scholar
  36. 36.
    Wong KKK, Tan LUL, Saddler JN (1988) Multiplicity of β-1,4-xylanase in microorganisms functions and applications. Microbiol Rev 52:305–317PubMedGoogle Scholar
  37. 37.
    Zamost BL, Nielsen HK, Starnes RL (1991) Thermostable enzymes for industrial applications. J Ind Microbiol 8:71–82Google Scholar
  38. 38.
    Zeikus JG, Lee C, Lee YE, Saha, BC (1991) Thermostable saccharidases: a new sources, uses and biodesign. In: Leatham GF, Himmel ME (eds) Enzymes in biomass conversion. American Chemical Society, Washington, pp 36–51Google Scholar

Copyright information

© Society for Industrial Microbiology 2004

Authors and Affiliations

  • Fabiana Fonseca Zanoelo
    • 1
  • Maria de Lourdes Teixeira de Moraes Polizeli
    • 1
  • Héctor Francisco Terenzi
    • 1
  • João Atílio Jorge
    • 1
  1. 1.Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão PretoUniversidade de São PauloRibeirão PretoBrasil

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