Skip to main content
SpringerLink
Log in

Cloning, Expression, and Purification of Xylanase Gene from Bacillus licheniformis for Use in Saccharification of Plant Biomass

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

The xylanase gene (xynA) of Bacillus licheniformis 9945A was cloned and expressed in Escherichia coli BL21(DE3) using pET-22b(+) as an expression vector. The recombinant xylanase enzyme was purified by ammonium sulfate precipitation, followed by single-step immobilized metal ion affinity chromatography with a 57.58-fold purification having 138.2 U/mg specific activity and recovery of 70.08 %. Molecular weight of the purified xylanase, 23 kDa, was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was stable for up to 70 °C with a broad pH range of 4–9 pH units. The enzyme activity was increased in the presence of metal ions especially Ca+2 and decreased in the presence of EDTA, indicating that the xylanase was a metalloenzyme. However, an addition of 1–4 % Tween 80, β-mercaptoethanol, and DTT resulted in the increase of enzyme activity by 51, 52, and 5 %, respectively. Organic solvents with a concentration of 10–40 % slightly decreased the enzyme activity. The xylanase enzyme possesses the ability of bioconversion of plant biomasses like wheat straw, rice straw, and sugarcane bagasse. Among the different tested biomasses, the highest saccharification percentage was observed with 1 % sugarcane bagasse after 72 h of incubation at 50 °C with 20 units of enzyme. The results suggest that recombinant xylanase can be used in the bioconversion of natural biomasses into simple sugars which could be further used for the production of biofuel.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Gupta, N., Reddy, V. S., Maiti, S., & Ghosh, A. (2000). Cloning, expression, and sequence analysis of the gene encoding the alkali-stable, thermostable endoxylanase from alkalophilic, mesophilic Bacillus sp. strain NG-27. Applied and Environmental Microbiology, 66, 2631–2635.

    Article  CAS  Google Scholar 

  2. Ninawe, S., Kapoor, M., & Kuhad, R. C. (2008). Purification and characterization of extracellular xylanase from Streptomyces cyaneus SN32. Bioresource Technology, 99, 1252–1258.

    Article  CAS  Google Scholar 

  3. Ghangas, G. S., Hu, Y. J., & Wilson, D. (1989). Cloning of a Thermomonospora fusca xylanase gene and its expression in Escherichia coli and Streptomyces lividans. Journal of Bacteriology, 171, 2963–2969.

    CAS  Google Scholar 

  4. Loera Corral, O., Villasenor-Ortega, F., Guevara-Gonzalez, R., & Torres-Pacheco, I. (2006). Xylanases. Advances in Agricultural and Food Biotechnology, 2006, 305–322.

    Google Scholar 

  5. Tachaapaikoon, C., Kyu, K. L., & Ratanakhanokchai, K. (2006). Purification of xylanase from alkaliphilic Bacillus sp. K-8 by using corn husk column. Process Biochemistry, 41, 2441–2445.

    Article  CAS  Google Scholar 

  6. Chidi, S. B., Godana, B., Ncube, I., Van Rensburg, E. J., Cronshaw, A., & Abotsi, E. K. (2008). Production, purification and characterization of celullase-free xylanase from Aspergillus terreus UL 4209. African Journal of Biotechnology, 7(21), 3939–3948.

    CAS  Google Scholar 

  7. Collins, T., Gerday, C., & Feller, G. (2005). Xylanases, xylanase families and extremophilic xylanases. FEMS Microbiology Reviews, 29, 3–23.

    Article  CAS  Google Scholar 

  8. Pollet, A., Sansen, S., Raedschelders, G., Gebruers, K., Rabijns, A., Delcour, J. A., & Courtin, C. M. (2009). Identification of structural determinants for inhibition strength and specificity of wheat xylanase inhibitors TAXI‐IA and TAXI‐IIA. FEBS Journal, 276, 3916–3927.

    Article  CAS  Google Scholar 

  9. Oakley, A. J., Heinrich, T., Thompson, C. A., & Wilce, M. C. (2003). Characterization of a family 11 xylanase from Bacillus subtillis B230 used for paper bleaching. Acta Crystallographica Section D: Biological Crystallography, 59, 627–636.

    Article  Google Scholar 

  10. Beg, Q., Kapoor, M., Mahajan, L., & Hoondal, G. (2001). Microbial xylanases and their industrial applications: a review. Applied Microbiology and Biotechnology, 56, 326–338.

    Article  CAS  Google Scholar 

  11. Subramaniyan, S., & Prema, P. (2002). Biotechnology of microbial xylanases: enzymology, molecular biology, and application. Critical Reviews in Biotechnology, 22, 33–64.

    Article  CAS  Google Scholar 

  12. Kamble, R. D., & Jadhav, A. R. (2012). Isolation, purification, and characterization of xylanase produced by a new species of Bacillus in solid state fermentation. International Journal of Microbiology, 2012(2), 1–8.

    Google Scholar 

  13. Korona, B., Korona, D., & Bielecki, S. (2006). Efficient expression and secretion of two co-produced xylanases from Aspergillus niger in Pichia pastoris directed by their native signal peptides and the Saccharomyces cerevisiae α-mating factor. Enzyme and Microbial Technology, 39, 683–689.

    Article  CAS  Google Scholar 

  14. Jamil, A., Nairn, S., Ahmad, S., & Ashraf, M. (2005). Production of industrially important enzymes using molecular approaches with special reference to xylanases and cellulases. Genetic Resources and Biotechnology, 2, 143.

    Google Scholar 

  15. Slapack, G. E., Russell, I., & Stewart, G. G. (1987). Thermophilic microbes in ethanol production.

  16. Screenath, H. K., Jeffries, W. T. (2000). Bioresour Technol., 253–60.

  17. Headon, D., & Walsh, G. (1994). The industrial production of enzymes. Biotechnology Advances, 12, 635–646.

    Article  CAS  Google Scholar 

  18. Schoemaker, H. E., Mink, D., & Wubbolts, M. G. (2003). Dispelling the myths biocatalysis in industrial synthesis. Science, 299, 1694–1697.

    Article  CAS  Google Scholar 

  19. Cazemier, A. E., Verdoes, J. C., Van Ooyen, A. J., & den Camp, H. J. O. (1999). Molecular and biochemical characterization of two xylanase-encoding genes from Cellulomonas pachnodae. Applied and Environmental Microbiology, 65, 4099–4107.

    CAS  Google Scholar 

  20. Srivastava, P., & Mukherjee, K. (2001). Cloning, characterization, and expression of xylanase gene from Bacillus lyticus in Escherichia coli and Bacillus subtilis. Preparative Biochemistry and Biotechnology, 31, 389–400.

    Article  CAS  Google Scholar 

  21. Rey, M. W., Ramaiya, P., Nelson, B. A., Brody-Karpin, S. D., Zaretsky, E. J., Tang, M., & Clausen, I. G. (2004). Complete genome sequence of the industrial bacterium Bacillus licheniformis and comparisons with closely related Bacillus species. Genome Biology, 5, 77.

    Article  Google Scholar 

  22. Kronstad, J., Schnepf, H., & Whiteley, H. (1983). Diversity of locations for Bacillus thuringiensis crystal protein genes. Journal of Bacteriology, 154, 419–428.

    CAS  Google Scholar 

  23. Cohen, S. N., Chang, A. C., & Hsu, L. (1972). Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proceedings of the National Academy of Sciences, 69, 2110–2114.

    Article  CAS  Google Scholar 

  24. Bimboim, H., & Doly, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Research, 7, 1513–1523.

    Article  Google Scholar 

  25. Laemmeli, U. (1970). Cleavage of structural B proteins during the assembly of the head to bacteriophage T4. Nature, 227, 680–685.

    Article  Google Scholar 

  26. Miller, G. (1959). Use of dinitrosalicylic acid reagent for determination reducing sugar. Analytical Chemistry, 31, 426–428.

    Article  CAS  Google Scholar 

  27. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254.

    Article  CAS  Google Scholar 

  28. de Moraes, L. M., Astolfi Filho, S., & Ulhoa, C. J. (1999). Purification and some properties of an α-amylase glucoamylase fusion protein from Saccharomyces cerevisiae. World Journal of Microbiology and Biotechnology, 15, 561–564.

    Article  Google Scholar 

  29. Grayson, M., Graham-Rowe, D., Sanderson, K., Martin, M., & Lynd, L. R. (2011). Woods. Nature, 2011, 1–24.

    Google Scholar 

  30. Turner, P., Mamo, G., & Karlsson, E. N. (2007). Potential and utilization of thermophiles and thermostable enzymes in biorefining. Microbial Cell Factories, 6, 1–23.

    Article  Google Scholar 

  31. Roayaei, M., & Galehdari, H. (2007). Cloning and expression of Thermus aquaticus DNA polymerase in Escherichia coli. Jundishapur Journal of Microbiology, 1, 1–5.

    Google Scholar 

  32. Gupta, N., Mehra, G., & Gupta, R. (2004). A glycerol-inducible thermostable lipase from Bacillus sp. medium optimization by a Plackett–Burman design and by response surface methodology. Canadian Journal of Microbiology, 50, 361–368.

    Article  CAS  Google Scholar 

  33. Bhasin, A., Razdan, K., Gupta, N., & Sethi, N. (2014). Production and Purification of alkali stable xylanase from Bacillus sp. International Journal of Current Microbiology and Applied Science, 3, 365–377.

    Google Scholar 

  34. Meera, P., Wallner, M., & Otis, T. S. (2011). Molecular basis for the high THIP/gaboxadol sensitivity of extrasynaptic GABAA receptors. Journal of Neurophysiology, 106, 2057–2064.

    Article  CAS  Google Scholar 

  35. Afzal, S., Saleem, M., Yasmin, R., Naz, M., & Imran, M. (2010). Pre and post cloning characterization of a β-1, 4-endoglucanase from Bacillus sp. Molecular Biology Reports, 37, 1717–1723.

    Article  CAS  Google Scholar 

  36. Knob, A., Beitel, S. M., Fortkamp, D., Terrasan, C. R. F., & Almeida, A. F. d. (2013). Production, purification, and characterization of a major Penicillium glabrum xylanase using Brewer’s spent grain as substrate. BioMed research international, 2013.

  37. Nakamura, S., Wakabayashi, K., Nakai, R., Aono, R., & Horikoshi, K. (1993). Purification and some properties of an alkaline xylanase from alkaliphilic Bacillus sp. strain 41M-1. Applied and Environmental Microbiology, 59, 2311–2316.

    CAS  Google Scholar 

  38. Chundakkadu Asha, P. (2011). Purification and biochemical characterization of xylanases from Bacillus pumilus and their potential for hydrolysis of polysaccharides. Fermentation Technology, 1, 1–8.

    Google Scholar 

  39. Akhavan Sepahy, A., Ghazi, S., & Akhavan Sepahy, M. (2011). Cost-effective production and optimization of alkaline xylanase by indigenous Bacillus mojavensis AG137 fermented on agricultural waste. Enzyme Research, 2011(2011), 1–9.

    Google Scholar 

  40. Huang, J., Wang, G., & Xiao, L. (2006). Cloning, sequencing and expression of the xylanase gene from a Bacillus subtilis strain B10 in Escherichia coli. Bioresource Technology, 97, 802–808.

    Article  CAS  Google Scholar 

  41. Khajeh, K., Khezre-Barati, S., & Nemat-Gorgani, M. (2001). Proteolysis of mesophilic and thermophilic α-amylases. Applied Biochemistry and Biotechnology, 94, 97–109.

    Article  CAS  Google Scholar 

  42. Gallardo, O., Diaz, P., & Pastor, F. J. (2004). Cloning and characterization of xylanase A from the strain Bacillus sp. BP-7: comparison with alkaline pI-low molecular weight xylanases of family 11. Current Microbiology, 48, 276–279.

    Article  CAS  Google Scholar 

  43. Chivero, E. T., Mutukumira, A. N., & Zvauya, R. (2001). Partial purification and characterization of a xylanase enzyme produced by a micro-organism isolated from selected indigenous fruits of Zimbabwe. Food Chemistry, 72, 179–185.

    Article  CAS  Google Scholar 

  44. Nair, S. G., Sindhu, R., & Shashidhar, S. (2008). Purification and biochemical characterization of two xylanases from Aspergillus sydowii SBS 45. Applied Biochemistry and Biotechnology, 149, 229–243.

    Article  CAS  Google Scholar 

  45. Heck, J. X., de Barros Soares, L. H., Hertz, P. F., & Ayub, M. A. Z. (2006). Purification and properties of a xylanase produced by Bacillus circulans BL53 on solid-state cultivation. Biochemical Engineering Journal, 32, 179–184.

    Article  CAS  Google Scholar 

  46. Bataillon, M., Cardinali, A. P. N., Castillon, N., & Duchiron, F. (2000). Purification and characterization of a moderately thermostable xylanase from Bacillus sp. strain SPS-0. Enzyme and Microbial Technology, 26, 187–192.

    Article  CAS  Google Scholar 

  47. Yuan, X., Wang, J., Yao, H., & Venant, N. (2005). Separation and identification of endoxylanases from Bacillus subtilis and their actions on wheat bran insoluble dietary fibre. Process Biochemistry, 40, 2339–2343.

    Article  CAS  Google Scholar 

  48. Khandeparker, R., Verma, P., & Deobagkar, D. (2011). A novel halotolerant xylanase from marine isolate Bacillus subtilis cho40: gene cloning and sequencing. New Biotechnology, 28, 814–821.

    Article  CAS  Google Scholar 

  49. Faulet, B. M., Niamke, S., Gonnety, J. T., & Kouame, L. P. (2006). Purification and biochemical properties of a new thermostable xylanase from symbiotic fungus, Termitomyces sp. African Journal of Biotechnology, 5, 273–282.

    CAS  Google Scholar 

  50. Faulet, B. M., Niamké, S., Gonnety, J. T., & Kouamé, L. P. (2006). Purification and characterization of two thermostable cellulase-free xylanases from workers of the termite Macrotermes subhyalinus (Isoptera: Termitidae). International Journal of Tropical Insect Science, 26, 108.

    Article  CAS  Google Scholar 

  51. Fialho, M., & Carmona, E. (2004). Purification and characterization of xylanases from Aspergillus giganteus. Folia Microbiologica, 49, 13–18.

    Article  CAS  Google Scholar 

  52. Dutta, T., Sengupta, R., Sahoo, R., Sinha Ray, S., Bhattacharjee, A., & Ghosh, S. (2007). A novel cellulase free alkaliphilic xylanase from alkali tolerant Penicillium citrinum: production, purification and characterization. Letters in Applied Microbiology, 44, 206–211.

    Article  CAS  Google Scholar 

  53. Do, T., Dam, T., & Quyen, D. (2009). Purification and biophysical characterization of xylanase from Aspergillus niger DSM 1957. Science Technology Journal of Agriculture and Rural Development, 6, 16–21.

    Google Scholar 

  54. Yang, Y., Zhang, W., Huang, J., Lin, L., Lian, H., Lu, Y., & Wang, S. (2010). Purification and characterization of an extracellular xylanase from Aspergillus niger C3486. African Journal of Microbiology Research, 4, 2249–2256.

    CAS  Google Scholar 

  55. Jeya, M., Thiagarajan, S., Lee, J.-K., & Gunasekaran, P. (2009). Cloning and expression of GH11 xylanase gene from Aspergillus fumigatus MKU1 in Pichia pastoris. Journal of Bioscience and Bioengineering, 108, 24–29.

    Article  CAS  Google Scholar 

  56. Chapla, D., Divecha, J., Madamwar, D., & Shah, A. (2010). Utilization of agro-industrial waste for xylanase production by Aspergillus foetidus MTCC 4898 under solid state fermentation and its application in saccharification. Biochemical Engineering Journal, 49, 361–369.

    Article  CAS  Google Scholar 

  57. Coughlan, M. (1991). Towards an understanding of the mechanism of action of main chain-hydrolyzing xylanases. Progress in Biotechnology, 7, 111–139.

    Article  Google Scholar 

  58. Dhabhai, R., Jain, A., & Chaurasia, S. P. (2012). Production of fermentable sugars by dilute acid pretreatment and enzymatic saccharification of three different lignocellulosic materials. International Journal of Chemistry Technology Research, 4, 1497–1502.

    CAS  Google Scholar 

  59. Mamo, G., Thunnissen, M., Hatti-Kaul, R., & Mattiasson, B. (2009). An alkaline active xylanase: insights into mechanisms of high pH catalytic adaptation. Biochimie, 91, 1187–1196.

    Article  CAS  Google Scholar 

  60. Liang, Y., Feng, Z., Yesuf, J., & Blackburn, J. W. (2010). Optimization of growth medium and enzyme assay conditions for crude cellulase produced by a novel thermophilic and cellulolytic bacterium, Anoxybacillus sp. 527. Applied Biochemistry and Biotechnology, 160, 1841–1852.

    Article  CAS  Google Scholar 

  61. Nagar, S., Gupta, V. K., Kumar, D., Kumar, L., & Kuhad, R. C. (2010). Production and optimization of cellulase-free, alkali-stable xylanase by Bacillus pumilus SV-85S in submerged fermentation. Journal of Industrial Microbiology & Biotechnology, 37, 71–83.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

Sincere gratitude is expressed to the Higher Education Commission (HEC), Islamabad, Pakistan, for funding this project as PhD research work for Ms. Asma Zafar.

Author information

Author notes

    Authors and Affiliations

    1. Institute of Industrial Biotechnology, Government College University, Lahore, 54000, Pakistan

      Asma Zafar, Muhammad Nauman Aftab, Zia ud Din, Irfana Iqbal, Anam Shahid & Ikram ul Haq

    2. Department of Environmental Science, Lahore College for Women University, Lahore, Pakistan

      Saima Aftab & Arifa Tahir

    Authors
    1. Asma Zafar
      View author publications

      You can also search for this author in PubMed Google Scholar

    2. Muhammad Nauman Aftab
      View author publications

      You can also search for this author in PubMed Google Scholar

    3. Zia ud Din
      View author publications

      You can also search for this author in PubMed Google Scholar

    4. Saima Aftab
      View author publications

      You can also search for this author in PubMed Google Scholar

    5. Irfana Iqbal
      View author publications

      You can also search for this author in PubMed Google Scholar

    6. Anam Shahid
      View author publications

      You can also search for this author in PubMed Google Scholar

    7. Arifa Tahir
      View author publications

      You can also search for this author in PubMed Google Scholar

    8. Ikram ul Haq
      View author publications

      You can also search for this author in PubMed Google Scholar

    Corresponding author

    Correspondence to Muhammad Nauman Aftab.

    Additional information

    Asma Zafar and Muhammad Nauman Aftab contributed equally to this work.

    Rights and permissions

    Reprints and permissions

    About this article

    Check for updates. Verify currency and authenticity via CrossMark

    Cite this article

    Zafar, A., Aftab, M.N., Din, Z.u. et al. Cloning, Expression, and Purification of Xylanase Gene from Bacillus licheniformis for Use in Saccharification of Plant Biomass. Appl Biochem Biotechnol 178, 294–311 (2016). https://doi.org/10.1007/s12010-015-1872-z

    Download citation

    • Received:

    • Accepted:

    • Published:

    • Issue Date:

    • DOI: https://doi.org/10.1007/s12010-015-1872-z

    Keywords

    Search

    Navigation