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Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
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Thermostable Xylanase from Marasmius sp.: Purification and Characterization

  • Received : 2005.10.20
  • Accepted : 2005.11.30
  • Published : 2006.01.31
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Abstract

We have screened 766 strains of fungi from the BIOTEC Culture Collection (BCC) for xylanases working in extreme pH and/or high temperature conditions, the so-called extreme xylanases. From a total number of 32 strains producing extreme xylanases, the strain BCC7928, identified by using the internal transcribed spacer (ITS) sequence of rRNA to be a Marasmius sp., was chosen for further characterization because of its high xylanolytic activity at temperature as high as $90^{\circ}C$. The crude enzyme possessed high thermostability and pH stability. Purification of this xylanase was carried out using an anion exchanger followed by hydrophobic interaction chromatography, yielding the enzyme with >90% homogeneity. The molecular mass of the enzyme was approximately 40 kDa. The purified enzyme retained broad working pH range of 4-8 and optimal temperature of $90^{\circ}C$. When using xylan from birchwood as substrate, it exhibits $K_m$ and $V_{max}$ values of $2.6{\pm}0.6\;mg/ml$ and $428{\pm}26\;U/mg$, respectively. The enzyme rapidly hydrolysed xylans from birchwood, beechwood, and exhibited lower activity on xylan from wheatbran, or celluloses from carboxymethylcellulose and Avicel. The purified enzyme was highly stable at temperature ranges from 50 to $70^{\circ}C$. It retained 84% of its maximal activity after incubation in standard buffer containing 1% xylan substrate at $70^{\circ}C$ for 3 h. This thermostable xylanase should therefore be useful for several industrial applications, such as agricultural, food and biofuel.

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References

  1. Bailey, M. J., Biely, P. and Poutanen, K. (1992) Interlaboratory testing of methods for assay of xylanase activity. J. Biotechnol. 23, 257-270. https://doi.org/10.1016/0168-1656(92)90074-J
  2. Bajpai, P. (1999) Application of enzymes in the pulp and paper industry. Biotechnol. Prog. 15, 147-157. https://doi.org/10.1021/bp990013k
  3. Biely, P. (1993) Biochemical aspects of the microbial hemicellulases; in Hemicelluloses and Hemicellulase, Coughlan, M. and Hazlewood, G. (eds.), pp. 29-51, Portland Press, London, U. K.
  4. 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. https://doi.org/10.1016/0003-2697(76)90527-3
  5. Buchan, A., Newell, S. Y., Moreta, J. I. and Moran, M. A. (2002) Analysis of internal transcribed spacer (ITS) regions of rRNA genes in fungal communities in a southeastern U.S. salt marsh. Microb. Ecol. 43, 329-340. https://doi.org/10.1007/s00248-001-1062-0
  6. Christakopoulos, P., Nerinckx, W., Kekos, D., Macris, B. and Claeyssens, M. (1996) Purification and characterization of two low molecular mass alkaline xylanases from Fusarium oxysporum F3. J. Biotechnol. 51, 181-189. https://doi.org/10.1016/0168-1656(96)01619-7
  7. Collin, T., Gerday, C. and Feller, G. (2005) Xylanases, xylanases families and extremophilic xylanases. FEMS Microbiol. Rev. 29, 3-23. https://doi.org/10.1016/j.femsre.2004.06.005
  8. Krisana, A., Rutchadaporn, S., Jarupan, G., Lily, E., Sutipa, T. and Kanyawim, K. (2005) Endo-1,4-${\beta}$-xylanase from Aspergillus cf. niger BCC14405 isolated in Thailand: purification, characterization and gene isolation. J. Biochem. Mol. Biol. 38, 17-23. https://doi.org/10.5483/BMBRep.2005.38.1.017
  9. Inagaki, K., Nakahira, K., Mukai, K., Tamura, T. and Tanaka, H. (1998) Gene cloning and characterization of an acidic xylanase from Acidobacterium capsulatum. Biosci. Biotechnol. Biochem. 62, 1061-1067. https://doi.org/10.1271/bbb.62.1061
  10. Hawksworth, D. L. (2001) The Magnitude of fungal diversity: the 1.5 million species estimated revisited. Mycol. Res. 105, 1422-1432. https://doi.org/10.1017/S0953756200002811
  11. Kalegoris, E., Christakopoulos, P., Kekos, D. and Macris B. J. (1998) Stydies on solid-state production of thermostable endoxylanases from Thermoascus aurantiacus. characterization of two isozymes. J. Biotechnol. 60, 155-163. https://doi.org/10.1016/S0168-1656(97)00186-7
  12. Kung, L. Jr., Treacher, R. J., Nauman, G. A., Smagala, A. M., Endres, K. M. and Cohen, M. A (2000) The effect of treating forages with fibrolytic enzymes on its nutritive value and lactation performance of dairy cows. J. Dairy Sci. 83, 115-122. https://doi.org/10.3168/jds.S0022-0302(00)74862-4
  13. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. https://doi.org/10.1038/227680a0
  14. Raj, K. C. and Chandra, T. S. (1996) Purification and characterization of xylanase from alkali-tolerant Aspergillus fischeri Fxn1. FEMS Microbio. Lett. 145, 457-461. https://doi.org/10.1111/j.1574-6968.1996.tb08616.x
  15. Rogalski, J., Oleszek, M. and Tokarzewska-Zadora, J. (2001) Purification and characterization of two endo-1, 4 beta-xylanase and a 3-xylosidase from Phlebia radiata. Acta. Microbiol. Pol. 50, 117-128.
  16. Romanowska, I., Polak, J., Janowska, K. and Bielecki, S. (2003) The application of fungal endoxylanase in bread-making. Commun. Agric. Appl. Biol. Sci. 68, 317-320.
  17. Romanowska, I., Polak, J. and Bielecki, S. (2005) Isolation and properties of Aspergillus niger IBT-90 xylanase for bakery. Appl. Microbiol. Biotechnol. 13, 1-7.
  18. Ruiz-Arribas, A., Fernandez, J. M., Sanchez, P., Garda, A. L. and Santamaria, R. I. (1995) Overproduction, purification, and biochemical characterization of a xylanase (Xys1) from Streptomyces halstedii JM8. Appl. Environ. Microbiol. 61, 2414-2419.
  19. Ryan, S. E., Nolan, K., Thompson, R., Gubitz, G. M., Savage, A. V. and Tuohy, M. G. (2003) Purification and characterization of a new low molecular weight endoxylanase from Penicillium capsularium. Enzyme Microb. Technol. 33, 775-785. https://doi.org/10.1016/S0141-0229(03)00176-5
  20. Sambrook, J. and Russell, D. W. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, New York, USA
  21. Silversides, F. G. and Bedford, M. R. (1999) Effect of pelleting temperature on the recovery and efficacy of a xylanase enzyme in wheat-based diets. Poult. Sci. 78, 1184-1190. https://doi.org/10.1093/ps/78.8.1184
  22. Sunna, A. and Antranikian, G. (1997) Xylanolytic enzymes from fungi and bacteria. Crit. Rev. Biotechnol. 17, 39-67. https://doi.org/10.3109/07388559709146606
  23. Tan, L. U. L., Wong, K. K. Y., Yu, E. K. C. and Saddler, J. N. (1985) Purification and characterization of two D-xylanases from Trichoderma harzianum. Enzyme. Microb. Technol. 7, 425-430. https://doi.org/10.1016/0141-0229(85)90041-9
  24. Velegraki, A., Kambouris, M., Kostourou, A., Chalevelakis, G. and Legakis, N. J. (1999) Rapid extraction of fungal DNA from clinical samples for PCR amplification. Food Technol. Biotechnol. 37, 69-73.
  25. Yeates, C., Gillings, M. R., Davison, A. D., Altavilla, N. and Veal, D. A. (1998) Methods for microbial DNA extraction from soil for PCR amplification. Biol. Proced. Online 1, 40-47. https://doi.org/10.1251/bpo6

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