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Purification and Biochemical Characterization of a Novel Thermo-stable Carboxymethyl Cellulase from Azorean Isolate Bacillus mycoides S122C

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Abstract

Bacillus mycoides S122C was identified as carboxymethyl cellulase (CMcellulase)-producing bacteria from the Azorean Bacillus collection (Lab collection), which was isolated from local soil samples. The bacteria was identified by 16S rRNA sequence and designated as B. mycoides S122C. NCBI blast analysis showed that the B. mycoides S122C 16S rRNA sequence has high identity compared to other B. mycoides strains. CMcellulase was purified from the culture filtrates using anion-exchange chromatography. After mono-Q purification, the protein folds and recovery were 13.7 and 0.76 %, respectively. SDS-PAGE analysis showed that the molecular weight of the purified CMcellulase protein was estimated to be about 62 kDa and that it was composed of a single subunit. MALDI-MS/MS analysis yielded each four peptides of the purified protein; it has identity to other cellulases. The purified CMcellulase showed high activity with CMcellulose followed by β-glucan as a substrate. Optimum temperature and pH for the purified CMcellulase activity were found to be at 50 °C and pH 7.0, respectively. The purified CMcellulase was stable with about 60 % activity in broad pH ranges from 5 to 10 and temperature of 40 to 60 °C. However, purified CMcellulase was stable at about 70 % at 70 °C and also stable overall at 78 % for surfactants. CMcellulase activity was inhibited by ions such as HgCl2, followed by CuSo4, FeCl2, and MnCl2, while CoCl2 activated CMcellulase activity. The purified CMcellulase activity was strongly inhibited by EDTA.

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References

  1. Yin, L. J., Lin, H. H., & Xiao, Z. R. (2010). Journal of Marine Science and Technology, 18, 466–471.

    Google Scholar 

  2. Coughlan, M. (1990). In W. Fogarty & C. Kely (Eds.), Microbial enzymes and biotechnology (pp. 1–36). London: Elsevier Applied Science.

    Chapter  Google Scholar 

  3. Kim, K. C., Yoo, S. S., Oh, Y. A., & Kim, S. J. (2003). Journal of Microbiology and Biotechnology, 13, 1–8.

    Google Scholar 

  4. Tomme, P., Warren, R. A., & Gilkes, N. R. (1995). Advances in Microbial Physiology, 37, 1–8.

    Article  CAS  Google Scholar 

  5. Kim, C. H. (1995). Applied and Environmental Microbiology, 61, 959–965.

    CAS  Google Scholar 

  6. Johnvesly, B., Virupakshi, S., Patil, G. N., & Ramalingam Naik, G. R. (2002). Journal of Microbiology and Biotechnology, 12, 153–156.

    CAS  Google Scholar 

  7. Lee, S. M., & Koo, Y. M. (2001). Journal of Microbiology and Biotechnology, 11, 229–233.

    CAS  Google Scholar 

  8. Subramaniyan, S., & Prema, P. (2000). FEMS Microbiology Letters, 183, 1–7.

    Article  CAS  Google Scholar 

  9. Doi, R. H. (2008). Annals of the New York Academy of Sciences, 1125, 267–279.

    Article  CAS  Google Scholar 

  10. Ariffin, H., Abdullah, N., Umi Kalsom, M. S., Shirai, Y., & Hassan, M. A. (2006). Journal of Engineering and Technology, 3, 47–53.

    Google Scholar 

  11. Bhat, M., & Bhat, S. (1997). Biotechnology Advances, 15, 583–620.

    Article  CAS  Google Scholar 

  12. Mandels, M. (1985). Biochemical Society Transactions, 13, 414–416.

    CAS  Google Scholar 

  13. Lee, Y. J., Kim, B. K., Lee, B. H., Jo, K. I., Lee, N. K., Chung, C. H., Lee, Y. C., & Lee, J. W. (2008). Bioresource Technology, 99, 378–386.

    Article  CAS  Google Scholar 

  14. Han, S. J., Yoo, Y. J., & Kang, H. S. (1995). Journal of Biological Chemistry, 270, 26012–26019.

    Article  CAS  Google Scholar 

  15. Singh, V. K., & Kumar, A. (1998). Biochemistry & Molecular Biology International, 45, 443–452.

    CAS  Google Scholar 

  16. Mawadza, C., Rajini, H. K., Zvauya, R., & Mattiasson, B. (2000). Journal of Biotechnology, 83, 177–187.

    Article  CAS  Google Scholar 

  17. Kotchoni, S. O., Gachomo, E. W., Omafuvbe, B. O., & Shonukan, O. O. (2006). International Journal of Agriculture and Biology, 8, 286–292.

    CAS  Google Scholar 

  18. Singh, J., Batra, N., & Sobti, R. C. (2004). Journal of Industrial Microbiology and Biotechnology, 31, 51–56.

    Article  CAS  Google Scholar 

  19. Yin, L. J., Huang, P. S., & Lin, H. H. (2010). Journal of Agricultural and Food Chemistry, 58, 9833–9837.

    Article  CAS  Google Scholar 

  20. Chen, P. J., Wei, T. C., Chang, Y. T., & Lin, L. P. (2004). Botanical Bulletin of Academia Sinica, 45, 11–18.

    Google Scholar 

  21. Sharma, P., Gupta, J. K., Vadhera, D. V., & Duae, D. I. C. (1990). Enzyme and Microbial Technology, 12, 132–137.

    Article  CAS  Google Scholar 

  22. Sarkar A. (1991). Geomicrobiology Journal, 9, 225–231.

    Google Scholar 

  23. Maki, M., Leung, K. T., & Qin, W. (2009). International Journal of Biological Sciences, 5(5), 500–516.

    Article  CAS  Google Scholar 

  24. Krieg, A. (1981). In M. P. Star, H. Stold, H. G. Truper, A. Balows, & H. G. Schlege (Eds.), The Prokaryotes, a handbook on habitats, isolation, and identification of bacteria (pp. 1743–1755). New York: Springer.

    Google Scholar 

  25. Wu, X. Y., Walker, M. J., Hornitzky, M., & Chin, J. (2006). Journal of Microbiological Methods, 64, 107–119.

    Article  CAS  Google Scholar 

  26. Liu, W., Marsh, T., Cheng, H., & Forney, L. (1997). Applied and Environmental Microbiology, 63, 4516–4522.

    CAS  Google Scholar 

  27. Thompson, J. D., Higgins, D. G., & Gibson, T. J. (1994). Nucleic Acids Research, 22, 4673–4680.

    Article  CAS  Google Scholar 

  28. Kumar, S., Tamura, K., & Nei, N. (1993). MEGA: Molecular evolutionary genetics analysis, version 1.01. University Park: The Pennsylvania State University.

    Google Scholar 

  29. Sampson, M. N., & Gooday, G. W. (1998). Microbiology, 144, 2189–2194.

    Article  CAS  Google Scholar 

  30. Miller, G. L., Blum, R., Glennon, W. E., & Burton, A. L. (1960). Analytical Biochemistry, 2, 127–132.

    Article  Google Scholar 

  31. Laemmli, U. K. (1970). Nature, 227, 680–685.

    Article  CAS  Google Scholar 

  32. Santos, R., da Costa, G., Franco, C., Gomes-Alves, P., Flammang, P., & Coelho, A. V. (2009). Marine Biotechnology, 11, 686–698.

    Article  CAS  Google Scholar 

  33. Chun, C. Z., Hur, S. B., & Kim, Y. T. (1997). Biochemistry & Molecular Biology International, 43, 241–249.

    CAS  Google Scholar 

  34. Endo, K., Hakamada, Y., Takizawa, S., & Kubota, H. (2001). Applied Microbiology and Biotechnology, 57, 109–116.

    Article  CAS  Google Scholar 

  35. Yoon, M. H., & Choi, W. Y. (2007). Journal of Microbiology and Biotechnology, 17, 1291–1299.

    CAS  Google Scholar 

  36. Nakamura, K., & Kitamura, K. (1983). Journal of Fermentation Technology, 61, 379–382.

    CAS  Google Scholar 

  37. Bischoff, K. M., Rooney, A. P., Li, X. L., Liu, S., & Hughes, S. R. (2006). Biotechnology Letters, 28, 1761–1765.

    Article  CAS  Google Scholar 

  38. Hakamada, Y., Endo, K., Takizawa, S., Kobayashi, T., Shirai, T., Yamane, T., & Ito, S. (2002). Biochimica et Biophysica Acta, 1570, 174–180.

    Article  CAS  Google Scholar 

  39. Ozaki, K., & Ito, S. (1991). Journal of General Microbiology, 37, 41–48.

    Article  Google Scholar 

  40. Saxena, S., Bahadur, J., & Varma, A. (1992). BioMetals, 5, 209–212.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by Fundo Regional Science and Technology (FRCT), Azores, Portugal. N. Balasubramanian gratefully acknowledges the post-doctoral grant M3.1.7/F/009A/2009 and also IBBA, Azores, Portugal, for financial support (M2.1.2/I/025/2008-RTF/2). We also acknowledge Mafalda Raposa, Vera Coelha, and Alexanda Ferrica for the technical assistance.

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Authors and Affiliations

  1. CIRN and Department of Biology, University of Azores, 9500-801, Ponta Delgada, Portugal

    Natesan Balasubramanian, Duarte Toubarro, Mário Teixeira & Nelson Simõs

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  1. Natesan Balasubramanian
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  2. Duarte Toubarro
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  3. Mário Teixeira
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  4. Nelson Simõs
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Correspondence to Natesan Balasubramanian.

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Balasubramanian, N., Toubarro, D., Teixeira, M. et al. Purification and Biochemical Characterization of a Novel Thermo-stable Carboxymethyl Cellulase from Azorean Isolate Bacillus mycoides S122C. Appl Biochem Biotechnol 168, 2191–2204 (2012). https://doi.org/10.1007/s12010-012-9929-8

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  • DOI: https://doi.org/10.1007/s12010-012-9929-8

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