Antonie van Leeuwenhoek

, Volume 69, Issue 3, pp 235–241 | Cite as

Purification and characterization of two different xylanases from the thermophilic actinomycete Microtetraspora flexuosa SIIX

  • Stephan Berens
  • Herwig Kaspari
  • Jobst-Heinrich Klemme


Two endoxylanases were isolated from the xylanolytic enzyme system of the thermophilic actinomycete Microtetraspora flexuosa SIIX, and purified by ammonium sulfate fractionation, DEAE-Sepharose chromatography, gel filtration on Sephacryl S 200 and fast protein liquid chromatography on Q-Sepharose. The molecular masses of xylanase I and II were 26.3 and 16.8 kDa, and isoelectric points were 8.4 and 9.45, respectively. optimal enzyme activities were obtained at 80° C and pH 6.0. The thermostability of both xylanases was greatly diminished during purification but could be restored by preincubation of the purified enzymes in the presence of xylan. The half-lives at 80° C were approximately 25 min. The kinetic constants of xylanases I and II determined with Remazol-brilliant-blue xylan were Vmax of 1537 and 353 μmol·min-1·mg protein-1 and K m values of 2.44 and 1.07 mg·ml-1, respectively. Purified xylanases utilized xylan as well as small oligosaccharides such as xylotriose as substrate. They did not exhibit xylobiase or debranching activities. The predominant products of arabinoxylan hydrolysis were xylobiose and xylotriose, the latter being hydrolysed to xylobiose and xylose upon further incubation. In addition, fragments containing arabinose side chains accumulated. The xylanases did not act on crystalline or amorphous cellulose indicating a possible application in biobleaching processes.

Key words

Microtetraspora thermophilic actinomycetes xylanases 


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  1. Bachmann, SL & McCarthy, AJ (1991) Purification and cooperative activity of enzymes constituting the xylan-degrading system of Thermomonospora fusca. Appl. Environ. Microbiol. 55: 1642–1644Google Scholar
  2. Ball, AS & McCarthy, AJ (1989) Production and properties of xylanases from actinomycetes. J. Appl. Bact. 66: 439–444Google Scholar
  3. Biely, P, Mislovicova, D & Toman, R (1988) Remazol-Brilliant-Blue-xylan: a soluble chromogenic substrate for xylanases. In: Wood, WA & Kellogg, ST (Eds) Methods in Enzymology, Vol. 160 (pp 536–541) Academic Press, San DiegoGoogle Scholar
  4. Biely, P, Puls, J & Schneider, H (1985) Acetyl-xylan-esterases in fungal cellulolytic systems. FEBS Lett. 186: 80–86Google Scholar
  5. Elegir, G, Szakas, G & Jeffries, TW (1994) Purification, characterization and substrate specificities of multiple xylanases from Streptomyces sp. strain B-12-2. Appl. Environ. Microbiol. 60: 2609–2615Google Scholar
  6. Éthier, JF, Harpin, S, Girard, C, Beaulieu, C, Déry, C & Brzezinsky, R (1994) Cloning of two xylanase genes from the newly isolated actinomycete Actinomadura sp. strain FC7 and characterization of the gene products. Can. J. Microbiol. 40: 362–368Google Scholar
  7. Fontana, JD, Gebara, M, Blumel, M, Schneider, H, MacKenzie, CR & Johnson, KG (1988) α-4-O-Methyl-D-glucuronidase component of xylanolytic complexes. In: Wood, WA & Kellogg, ST (Eds) Methods in Enzymology, Vol. 160 (pp 560–571). Academic Press, San DiegoGoogle Scholar
  8. Fontes, CMGA, Hall, J, Hirst, BH, Hazlewood, GP & Gilbert, HJ (1995) The resistance of cellulases and xylanases to proteolytic inactivation. Appl. Microbiol. Biotechnol. 43: 52–57PubMedGoogle Scholar
  9. Greiner-Mai, E, Kroppenstedt, RM, Korn-Wendisch, F & Kutzner, HJ (1987) Morphological and biochemical characterization and emended descriptions of thermophilic actinomycetes species. System. Appl. Microbiol. 9: 97–109Google Scholar
  10. Grüninger, H & Fiechter, A (1986) A novel highly thermostable D-xylanase. Enz. Microb. Technol. 8: 309–314Google Scholar
  11. Holtz, C, Kaspari, H & Klemme, JH (1991) Production and properties of xylanases from thermophilic actinomycetes. Antonie van Leeuwenhoek 59: 1–7PubMedGoogle Scholar
  12. Irwin, D, Jung, ED & Wilson, DB (1994) Characterization and sequence of a Thermomonospora fusca xylanase. Appl. Environ. Microbiol. 60: 763–770PubMedGoogle Scholar
  13. Kluepfel, D, Vats-Metha, S, Aumont, F, Shareck, F & Molosoli, R (1990) Purification and characterization of a new xylanase (xylanase B) produced by Streptomyces lividans 66. Biochem. J. 267: 45–50PubMedGoogle Scholar
  14. Kormelink, FJM & Voragen, AGJ (1992) Degradation of different [(glucurono)arabino] xylans by a combination of purified xylan-degrading enzymes. Appl. Microbiol. Biotechnol. 38: 688–695Google Scholar
  15. Lowry, OH, Rosebourgh, NJ, Farr, AL & Randall, RJ (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265–275PubMedGoogle Scholar
  16. Marui, M, Nakanishi, K & Yasui, T (1985) Purification and properties of three types of xylanases induced by methyl-β-xyloside from Streptomyces sp. Agric. Biol. Chem. 49: 3399–3407Google Scholar
  17. Milagres, AMF & Prade, RA (1994) Production of xylanases from Penicillium janthinellum and its use in the recovery of cellulosic textile fibers. Enz. Microb. Technol. 16: 627–632Google Scholar
  18. Miller, GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31: 426–428Google Scholar
  19. Nakanishi, K, Marui, M & Yasui, T (1992) Comparison of xylan and methyl β-xyloside-induced xylanases from Streptomyces sp. J. Ferment. Bioeng. 74: 392–394Google Scholar
  20. Nonomura, H & Ohara, Y (1971) Distribution of actinomycetes in soil. J. Ferment. Technol. 49: 904–912Google Scholar
  21. Ristroph, DL & Humphrey, AE (1985) Kinetic characterization of the extracellular xylanases of Thermomonospora sp. Biotechnol. Bioeng. 28: 832–836Google Scholar
  22. Skoog, K & Hahn-Hägerdahl, B (1987) Xylose fermentation. Enz. Microb. Technol. 10: 66–80Google Scholar
  23. Tanaka, T, Shimomura, Y, Himejima, M, Taniguchi, M & Oi, S (1986) Characterization of xylan-utilizing anaerobes from mesophilic and thermophilic methane sludge and their xylan degrading enzymes. Agric. Biol. Chem. 50: 2185–2192Google Scholar
  24. Viikari, L, Kantelinen, A, Sundquist, J & Linko, M (1994) Xylanases in bleaching: from an idea to the industry. FEMS Microbiol. Rev. 13: 335–350Google Scholar
  25. Wilkie, K (1979) The hemicelluloses of grasses and cereals. Adv. Carbohydr. Chem. Biochem. 10: 215–262Google Scholar
  26. Wong, KKJ, Tan, LUL & Saddler, JN (1988) Multiplicity of β-1,4-xylanase in microorganisms: functions and applications. Microbiol. Rev. 52: 305–317PubMedGoogle Scholar
  27. Wood, TM (1988) Preparation of crystalline, amorphous, and dyed cellulase substrates. In: Wood, WA & Kellogg, ST (Eds) Methods in Enzymology, Vol. 160 (pp 19–25) Academic Press, San DiegoGoogle Scholar

Copyright information

© Kluwer Academic Publishers 1996

Authors and Affiliations

  • Stephan Berens
    • 1
  • Herwig Kaspari
    • 1
  • Jobst-Heinrich Klemme
    • 1
  1. 1.Institut für Mikrobiologie und Biotechnologie der Universität BonnBonnGermany

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