Applied Biochemistry and Biotechnology

, Volume 170, Issue 1, pp 119–130 | Cite as

Thermostable and Alkalistable Endoxylanase of the Extremely Thermophilic Bacterium Geobacillus thermodenitrificans TSAA1: Cloning, Expression, Characteristics and Its Applicability in Generating Xylooligosaccharides and Fermentable Sugars



Xylanase encoding gene (1,224 bp) from Geobacillus thermodenitrificans was cloned in pET28a (+) vector and successfully expressed in Escherichia coli BL21 (DE3). The deduced amino acid sequence analysis revealed homology with that of glycosyl hydrolase (GH) 10 family with a high molecular mass (50 kDa). The purified recombinant xylanase is optimally active at pH 9.0 and 70 °C with T1/2 of 10 min at 80 °C, and retains greater than 85 % activity after exposure to 70 °C for 180 min. The enzyme liberates xylose as well as xylooligosaccharides from birchwood xylan and agro-residues, and therefore, this is an endoxylanase. The xylan hydrolytic products (xylooligosaccharides, xylose, and xylobiose) find application as prebiotics and in the production of bioethanol. The xylanase being thermostable and alkalistable, it has released chromophores and phenolics from the residual lignin of pulps, suggesting its utility in mitigating chlorine requirement in pulp bleaching.


Geobacillus thermodenitrificans Thermo-alkalistable xylanase Pulp pre-bleaching Xylooligosaccharides Prebiotics 

Supplementary material

12010_2013_174_MOESM1_ESM.doc (40 kb)
Supplementary Fig. 1Nucleotide and deduced amino acid sequences of the G. thermodenitrificans xylanase gene. The underlined regions I (I V A E N V M K), II (R F H T L V W H), III (D V V N E), IV (L Y I N D Y N), V (I G H Q S H I), VI (I T E L D V), VII (T F W G I A D N H T W), and VIII (D Y I K V A F Q T A) denote highly conserved GH10 xylanases. Glu187, Asp230, and Glu293 are crucial catalytic residues. (DOC 40 kb)
12010_2013_174_MOESM2_ESM.doc (237 kb)
Supplementary Fig. 2Secondary structure of rXyl-gtd was proposed using ESPript 2.2 software. The structure is based on the available template of xylanase from 1HIZ chain A of G. stearothermophilus. Symbols α, β, and TT denote the helix, sheets, and turns, respectively. (DOC 237 kb)
12010_2013_174_MOESM3_ESM.doc (644 kb)
Supplementary Fig.3Electrophoretic analysis of the rXyl-gtd using 15 % SDS-PAGE. a Purified rXyl-gtd of 50 kDa; M, standard protein markers; lanes 1 and 2 show eluted proteins with 200 and 250 mM imidazole; b zymogram analysis of purified rXyl-gtd using Congo red. (DOC 644 kb)


  1. 1.
    Collins, T., Gerday, C., & Feller, G. (2005). FEMS Microbiology Reviews, 29, 3–23.CrossRefGoogle Scholar
  2. 2.
    Bazzicalupo, M., & Fani, R. (1995). In: Methods in molecular biology, species diagnostic protocols: PCR and other nucleic acids methods. (Eds. Clapp, J.P.), pp. 155–77. Totowa: Humana Press.Google Scholar
  3. 3.
    Verma, D., & Satyanarayana, T. (2012). Bioresource Technology, 107, 333–338.CrossRefGoogle Scholar
  4. 4.
    Archana, A., & Satyanarayana, T. (1997). Enzyme and Microbial Technology, 21, 12–17.CrossRefGoogle Scholar
  5. 5.
    Miller, G. L. (1959). Anaytical Chemistry, 31, 426–428.CrossRefGoogle Scholar
  6. 6.
    Patel, R. N., Grabski, A. C., & Jeffries, T. W. (1993). Applied Microbiology and Biotechnology, 39, 405–412.CrossRefGoogle Scholar
  7. 7.
    Lambert, C., Leonard, N., De Bolle, X., & Depiereux, E. (2002). Bioinformatics, 18, 1250–1256.CrossRefGoogle Scholar
  8. 8.
    Gerasimova, J., & Kuisiene, N. (2012). Microbiology, 81, 418–424.CrossRefGoogle Scholar
  9. 9.
    Saksono, B., & Sukmarini, L. (2010). HAYATI Journal of Biosciences, 17, 189–197.CrossRefGoogle Scholar
  10. 10.
    Canakci, S., Cevher, Z., Inan, K., Tokgoz, M., Bahar, F., Kacagan, M., et al. (2012). World Journal of Microbiology and Biotechnology, 28, 1981–1988.CrossRefGoogle Scholar
  11. 11.
    Mamo, G., Hatti-Kaul, R., & Mattiasson, B. (2007). Extremophiles, 11, 169–177.CrossRefGoogle Scholar
  12. 12.
    Guo, B., Chen, X. L., Sun, C. Y., Zhou, B. C., & Zhang, Y. Z. (2009). Applied Microbiology and Biotechnology, 84, 1107–1115.CrossRefGoogle Scholar
  13. 13.
    Sinnot, M. L. (1990). Chemical Reviews, 90, 1171–1202.CrossRefGoogle Scholar
  14. 14.
    McCarter, J. D., & Withers, S. G. (1994). Current Opinion in Structure Biology, 4, 885–892.CrossRefGoogle Scholar
  15. 15.
    Shi, P., Tian, J., Yuan, T., Liu, X., Huang, H., et al. (2011). Applied and Environmental Microbiology, 76, 3620–3624.CrossRefGoogle Scholar
  16. 16.
    Ko, E. P., Akatsuka, H., Moriyama, H., Shinmyo, A., Hata, Y., Kastube, Y., et al. (1992). Journal of Biochemistry, 288, 117–121.Google Scholar
  17. 17.
    Coughlan, M., & Visser, J. (1992). In J. Visser, G. Beldman, M. A. K. Someren, & A. G. J. Voragen (Eds.), Xylans and xylanases (pp. 111–140). Amsterdam: Elsevier.Google Scholar
  18. 18.
    Paes, G., Berrin, J. G., & Beaugrand, J. (2012). Advances in Biotechnology, 30, 564–592.CrossRefGoogle Scholar
  19. 19.
    Jalal, A., Rashid, N., Rasool, N., & Akhtar, M. (2009). Journal of Bioscience and Bioengineering, 107, 360–365.CrossRefGoogle Scholar
  20. 20.
    Hyeong, H. L., Lim, P. O., & Lee, Y. E. (2007). Journal of Microbiology and Biotechnology, 17, 29–36.Google Scholar
  21. 21.
    Son-Ng, I., Li, C. W., Yeh, Y., Chen, P. T., Chir, J. L., Ma, C. H., et al. (2009). Extremophiles, 13, 425–435.CrossRefGoogle Scholar
  22. 22.
    Chang, W. S., Tsai, C. L., & Tseng, M. J. (2004). Biochemical and Biophysical Research Communications, 319, 1017–1025.CrossRefGoogle Scholar
  23. 23.
    Cheng, Y. F., Yang, C. H., & Liu, W. H. (2005). Enzyme and Microbial Technology, 37, 541–546.CrossRefGoogle Scholar
  24. 24.
    Gupta, N., Vohra, R. M., & Hoondal, G. S. (1992). Biotechnology Letters, 14, 1045–1046.CrossRefGoogle Scholar
  25. 25.
    Bajaj, B. K., Razdan, K., & Sharma, A. (2010). Indian Journal of Chemical Technology, 17, 375–380.Google Scholar
  26. 26.
    Zhang, G., Mao, L., Zhao, Y., Xue, Y., & Ma, Y. (2010). Biotechnology Letters, 32, 1915–1920.CrossRefGoogle Scholar
  27. 27.
    Khasin, A., Alchanati, I., & Shoham, Y. (1993). Applied and Environmental Microbiology, 59, 1725–1730.Google Scholar
  28. 28.
    Shrinivas, D., Savitha, G., & Naik, G. R. (2010). Applied Biochemistry and Biotechnology, 162, 2049–2057.CrossRefGoogle Scholar
  29. 29.
    Viikari, L., Ranua, M., Kantelinen, A., Sunduist, J., & Linko, M. (1986). In: Proc 3rd Int Conf Biotechnology in the Pulp and Paper Industry, STFI, Stockholm, Sweden, pp.67–9.Google Scholar
  30. 30.
    Kulkarni, N., & Rao, N. (1996). Journal of Biotechnology, 51, 167–173.CrossRefGoogle Scholar
  31. 31.
    Zhang, G. M., Huang, J., Huang, G. R., Ma, L. X., & Zhang, X. E. (2007). Applied Microbiology and Biotechnology, 74, 339–346.CrossRefGoogle Scholar
  32. 32.
    Liu, W., Shi, P., Chen, Q., Yang, P., Wang, G., Wang, Y., et al. (2010). Applied Biochemistry and Biotechnology, 162, 1–12.CrossRefGoogle Scholar
  33. 33.
    Yin, L., Lin, H., Chiang, Y., & Jiang, S. (2010). Journal of Agriculture & Food Chemistry, 58, 557–562.CrossRefGoogle Scholar
  34. 34.
    Gupta, S., Bhushan, B., & Hoondal, G. S. (2000). Journal of Applied Microbiology, 88, 325–334.CrossRefGoogle Scholar
  35. 35.
    Kamble, R. D., & Jadhav, A. R. (2012). ISRN Biotechnology, 2013, 1–5.CrossRefGoogle Scholar
  36. 36.
    Salama, M. A., Ismail, K. M. I., Amany, H. A., Abo, E.-L., & Genweely, N. S. I. (2008). International Journal of Botany, 4, 41–48.CrossRefGoogle Scholar
  37. 37.
    Lu, F., Lu, M., Lu, Z., Bie, X., Zhao, H., & Wang, Y. (2008). Bioresource Technology, 99, 5938–5941.CrossRefGoogle Scholar
  38. 38.
    Cardoso, O. A. V., & Filho, E. X. F. (2003). FEMS Microbiology Letters, 223, 309–314.CrossRefGoogle Scholar
  39. 39.
    Wu, S., Liu, B., & Zhang, X. (2006). Applied Microbiology and Biotechnology, 72, 1210–1216.CrossRefGoogle Scholar
  40. 40.
    Do, T. T., & Quyen, D. T. (2010). Middle East Journal of Scientific Research, 6, 392–397.Google Scholar
  41. 41.
    Torronen, A., Mach, R. L., Messner, R., Gonzalez, R., Kakkinen, N., Harkki, A., et al. (1992). Biotechnology (NY), 10, 1461–1465.CrossRefGoogle Scholar
  42. 42.
    Khandeparkar, R. D. S., & Bhosle, N. B. (2006). Enzyme and Microbial Technology, 4, 732–742.CrossRefGoogle Scholar
  43. 43.
    Gruppen, H., Hamer, R. J., & Voragen, A. G. J. (1992). Journal of Cereal Science, 16, 53–67.CrossRefGoogle Scholar
  44. 44.
    Kormelink, F. J. M., & Voragen, A. G. J. (1993). Applied Microbiology and Biotechnology, 38, 688–695.CrossRefGoogle Scholar
  45. 45.
    Jiang, Z. Q., Deng, W., Zhu, Y. P., Li, L. T., Sheng, Y. J., & Hayashi, K. (2004). Journal of Molecular Catalysis B Enzymatic, 27, 207–213.CrossRefGoogle Scholar
  46. 46.
    Ratanakhanokchai, K., Kyu, K. L., & Tanticharoen, M. (1999). Applied and Environmental Microbiology, 65, 694–697.Google Scholar
  47. 47.
    Damiano, V. B., Bocchini, D. A., Gomes, E., & Da Silva, R. (2003). World Journal of Microbiology and Biotechnology, 19, 139–144.CrossRefGoogle Scholar
  48. 48.
    Choudhury, B., Sunita, C., Singh, S. N., & Ghosh, P. (2006). World Journal of Microbiology and Biotechnology, 22, 283–288.CrossRefGoogle Scholar
  49. 49.
    Kaur, A., Mahajan, R., Singh, A., Garg, G., & Sharma, J. (2010). Bioresource Technology, 101, 9150–9155.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Digvijay Verma
    • 1
  • Ashima Anand
    • 1
  • T. Satyanarayana
    • 1
  1. 1.Department of MicrobiologyUniversity of Delhi South CampusNew DelhiIndia

Personalised recommendations