Applied Biochemistry and Biotechnology

, Volume 164, Issue 1, pp 77–88 | Cite as

Purification and Characterization of Chitinase from Paenibacillus sp. D1

  • Anil Kumar Singh
  • Hari S. ChhatparEmail author


A 56.56-kDa extracellular chitinase from Paenibacillus sp. D1 was purified to 52.3-fold by ion exchange chromatography using SP Sepharose. Maximum enzyme activity was recorded at pH 5.0 and 50 °C. MALDI-LC-MS/MS analysis identified the purified enzyme as chitinase with 60% similarity to chitinase Chi55 of Paenibacillus ehimensis. The activation energy (E a) for chitin hydrolysis and temperature quotient (Q 10) at optimum temperature was found to be 19.14 kJ/mol and 1.25, respectively. Determination of kinetic constants k m, V max, k cat, and k cat/k m and thermodynamic parameters ΔH*, ΔS*, ΔG*, ΔG*E–S, and ΔG*E–T revealed high affinity of the enzyme for chitin. The enzyme exhibited higher stability in presence of commonly used protectant fungicides Captan, Carbendazim, and Mancozeb compared to control as reflected from the t 1/2 values suggesting its applicability in integrated pest management for control of soil-borne fungal phytopathogens. The order of stability of chitinase in presence of fungicides at 80 °C as revealed from t 1/2 values and thermodynamic parameters E a(d) (activation energy for irreversible deactivation), ΔH*, ΔG*, and ΔS* was: Captan > Carbendazim > Mancozeb > control. The present study is the first report on thermodynamic and kinetic characterization of chitinase from Paenibacillus sp. D1.


Chitinase Fungicides IPM Kinetics Paenibacillus Thermodynamics 



The work was supported by UGC/DRS Phase-II, New Delhi, India.


  1. 1.
    Gohel, V., Singh, A., Maisuria, V., Phadnis, A., & Chhatpar, H. S. (2006). African Journal of Biotechnology, 5, 54–72.Google Scholar
  2. 2.
    Nawani, N. N., Prakash, D., & Kapadnis, B. P. (2010). World Journal of Microbiology & Biotechnology, 26, 1509–1517.CrossRefGoogle Scholar
  3. 3.
    Aam, B. B., Heggset, E. B., Norberg, A. L., Sørlie, M., Vårum, K. M., & Eijsink, V. G. H. (2002). Marine Drugs, 8, 1482–1517.CrossRefGoogle Scholar
  4. 4.
    Balsubramanium, N., Juliet, G. A., Srikalaivani, P., & Lalithakumari, D. (2003). Canadian Journal of Microbiology, 49, 263–268.CrossRefGoogle Scholar
  5. 5.
    Rhishipal, R., & Philip, R. (1998). Bioresource Technology, 65, 255–256.CrossRefGoogle Scholar
  6. 6.
    Bhattacharya, D., Nagpure, A., & Gupta, R. K. (2007). Critical Reviews in Biotechnology, 27, 21–28.CrossRefGoogle Scholar
  7. 7.
    von der Weid, I., Alviano, D. S., Santos, A. L. S., Soares, R. M. A., Alviano, C. S., & Seldin, L. (2003). Journal of Applied Microbiology, 95, 1143–1151.CrossRefGoogle Scholar
  8. 8.
    Budi, S. W., van Tuinen, D., Arnould, C., Dumas-Gaudut, E., Gianinazzi-Pearson, V., & Gianinazzi, S. (2000). Applied Soil Ecology, 15, 191–199.CrossRefGoogle Scholar
  9. 9.
    Singh, A. K., Ghodke, I., & Chhatpar, H. S. (2009). Journal of Environmental Management, 91, 358–362.CrossRefGoogle Scholar
  10. 10.
    Vieille, C., & Zeikus, G. J. (2001). Microbiology and Molecular Biology Reviews, 65, 1–43.CrossRefGoogle Scholar
  11. 11.
    Singh, A. K., Mehta, G., & Chhatpar, H. S. (2009). Letters in Applied Microbiology, 49, 708–714.CrossRefGoogle Scholar
  12. 12.
    Vyas, P., & Deshpande, M. V. (1989). The Journal of General and Applied Microbiology, 35, 343–350.CrossRefGoogle Scholar
  13. 13.
    Laemmli, U. K. (1970). Nature, 277, 680–685.CrossRefGoogle Scholar
  14. 14.
    Sambrook, J., & Russell, D. W. (2001). Molecular cloning: A laboratory manual. New York: Cold Spring Harbor Laboratory.Google Scholar
  15. 15.
    Dixon, M., & Webb, E. C. (1979). Enzymes. New York: Academic.Google Scholar
  16. 16.
    Eyring, H., & Stearn, A. E. (1939). Chemical Reviews, 24, 253–270.CrossRefGoogle Scholar
  17. 17.
    Laidler, K. J., & Peterman, B. F. (1979). Methods in Enzymology, 63, 234–257.CrossRefGoogle Scholar
  18. 18.
    Jensen, M. H., Malter, A. J., Jensen, M. H., & Malter, A. J. (1995). Protected agriculture: A global review (pp. 75–76). Washington DC: World Bank.Google Scholar
  19. 19.
    Guetsky, R., Shtienberg, D., Elad, Y., Fischer, E., & Dinoor, A. (2002). Phytopathology, 92, 976–985.CrossRefGoogle Scholar
  20. 20.
    Muhammad, R., Raheela, P., Muhammad, R. J., Habibullah, N., & Muhammad, H. R. (2007). Enzyme and Microbial Technology, 41, 558–564.CrossRefGoogle Scholar
  21. 21.
    Jacobsen, B. J., Zidack, N. K., & Larson, B. J. (2004). Phytopathology, 94, 1272–1275.CrossRefGoogle Scholar
  22. 22.
    Kiewnick, S., Jacobsen, B. J., Braun-Kiewnick, A., Eckhoff, J. L. A., & Bergman, J. W. (2001). Plant Disease, 85, 718–722.CrossRefGoogle Scholar
  23. 23.
    Someya, N., Tsuchiya, K., Yoshida, T., Tsujimoto-Noguchi, M., & Sawada, H. (2007). Biocontrol Science and Technology, 17, 21–31.CrossRefGoogle Scholar
  24. 24.
    D’Amico, S., Marx, J.-C., Gerday, C., & Feller, G. (2003). The Journal of Biological Chemistry, 278, 7891–7896.CrossRefGoogle Scholar

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© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.Department of Microbiology and Biotechnology Centre, Faculty of ScienceThe Maharaja Sayajirao University of BarodaVadodaraIndia

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