Abstract
The purpose of the research was to study the purification and partial characterization of antifungal alkaline chitinase from a newly isolated Citrobacter freundii haritD11. The enzyme was purified in a three-step procedure involving ammonium sulfate precipitation, dialysis, and Sephadex G-100 gel filtration chromatography. The enzyme was shown to have a relative high molecular weight of 64 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and was purified 7.3-fold with a yield of 18.8 %. It was most active at 35 °C, pH 8.0, with colloid chitin as substrate and was very stable at alkaline pH contradicting the characteristic that most of the bacterial chitinases are active at acidic pH. Further, the purified chitinase exhibited remarkable antifungal activity against pathogenic fungi Aspergillus flavus MTCC 2798 and Aspergillus niger MTCC 9652 showing diametric inhibition zones of 27 mm and 21 mm, respectively.
Similar content being viewed by others
References
Souza, C. P., AlmeidaColwell, B. C., Colwell, R. R., & Rivera, I. N. G. (2011). Marine Biotechnology, 13, 823–830.
Hamid, R., Minhaj, A. K., Mahboob, A., Malik Mobeen, A., Malik Zainul, A., Javed, M., et al. (2013). Journal of Pharmaceutical Bioallied Science, 5, 21–29.
O'keefe, D. M., Owens, J. M., & Chynoweth, D. P. (1996). Bioresource Technology, 58, 265–272.
Se-Kwon (2013) Marine Nutraceuticals: Prospects and Perspectives. CRC Press, pp. 315–319.
Wang, S. L., Lin, T. Y., Yen, Y. H., Liao, H. F., & Chen, Y. J. (2006). Carbohydrate Research, 341, 2507–2515.
Sabu, A. (2003). Indian Journal of Biotechnology, 2, 334–341.
Miller, G. L. (1959). Analytical Biochemistry, 3, 426–428.
Lowry, O. H., Rosenbrough, N. J., Farr, A. L., & Randall, J. R. (1951). Journal of Biological Chemistry, 193, 265–275.
Jakoby, W. B. (1971). Enzyme purification and related techniques. Methods in Enzymology, vol XXII (pp. 273–315). USA: Academic Press.
Rosenberg, I. M. (1996). Protein analysis and purification: benchtop techniques (pp. 119–125). Boston: Birkhauser.
Uma, C., Balakrishnan, S., Duraisamy, G., Manokaran, K., Ganesan, R., & Chinthamani, A. (2012). Journal of Advanced Scientific Research, 3, 25–29.
Narayana Kolla, J. P., & Vijayalakshmi, M. (2009). Braz. Journal of Microbiology, 40, 725–733.
Guo, S. H., Chen, J. K., & Lee, W. C. (2004). Enzyme and Microbial Technology, 35, 550–556.
Laemmli, U. K. (1970). Nature, 227, 680–685.
Nawani, N. N., Kapadnis, B. P., Das, A. D., Rao, A. S., & Mahajan, S. K. (2002). Journal of Applied Microbiology, 93, 965–975.
Rabeeth, M., Anitha, A., & Geetha, S. (2011). Pakistan Journal of Biological Sciences, 14, 788–797.
Yamaoka, H., Hayashi, H., Karita, S., Kimura, T., Sakka, K., & Ohmiya, K. (1999). Journal of Bioscience and Bioengineering, 88, 328–330.
Hiraga, K., Shou, L., Kitazawa, M., Takahashi, S., Shimada, M., Sato, R., et al. (1997). Bioscience, Biotechnology, and Biochemistry, 61, 174–176.
Dahiya, N., Tewari, R., Tiwari, R. P., & Hoondal, G. S. (2005). World Journal of Microbiology and Biotechnology, 21, 1611–1616.
Agrios George N. (2005) Plant Pathology: 5th ed. Elsevier Academic. pp. 922.
Ueno, H., Miyashita, K., Swada, Y., & Oba, Y. (1990). Journal of General and Applied Microbiology, 36, 377–392.
Reyes-Ramírez, A., Escudero-Abarca, B. I., Aguilar-Uscanga, G., Hayward-Jones, P. M., & Eleazar Barbozacorona, J. (2004). Journal of Food Science, 69, M131–M134.
Haggag Wafaa, M., & Abdallh Elham, G. (2012). Journal of Microbiology Research, 2, 145–151.
Annamalai, N., Mayavan, V. R., Shanmugam, V., & Thangavel, B. (2011). Annals of Microbiology, 61, 801–807.
Te Lien, S., Shwu-Tzy, W., Shin-Tsung, Y., & Jui-Rze, T. (2007). Korean Journal Chemical Engineering, 24, 806–811.
De-hui, D., Wei-lian, H., Huang, G.-r., & Li, W. (2011). African Journal of Biotechnology, 10, 2476–2485.
Nawani, N. N., & Kapadnis, B. P. (2003). Indian Journal of Experimental Biology, 41, 248–254.
Singh, A. K., & Chhatpar, H. S. (2011). Applied Biochemistry Biotechnology, 164, 77–88.
Yong, T., Hong, J., Zhangfu, L., Li, Z., Xiuqiong, D., Ke, T., et al. (2005). Annals of Microbiology, 55, 213–218.
Frankowski, J., Lorito, M., Scala, F., Schmid, R., Berg, G., & Bahl, H. (2001). Archives of Microbiology, 176, 421–426.
Harty, D. W. S., Chen, Y., Simpson, C. L., Berg, T., Cook, S. L., Mayo, J. A., et al. (2004). Biochemical and Biophysical Research Communications, 319, 439–447.
Kim Kyoung, J., Yang Yong, J., & Kim Jong, G. (2003). Journal of Biochemistry and Molecular Biology, 36, 185–189.
Vaidya, R., Vyas, P., & Chhatpar, H. S. (2003). Enzyme Microbiology Technology, 33, 92–96.
Murata, T., Amarume, S., Hattori, T., Tokuyama, S., Tokuyasu, K., Kawagishi, H., et al. (2005). Biochemical and Biophysical Research Communications, 336, 514–520.
Wang, S. Y., Moyne, A. L., Thottappilly, G., Wu, S. J., Locy, R. D., & Singh, N. K. (2001). Enzyme Microbiology Technology, 28, 492–498.
Morimoto, K. S., Karita, T., Kimura, T., Sakka, K., & Ohmiya, K. (1997). Journal of Bacteriology, 179, 7306–7314.
Lee, H. S., Han, D. S., Choi, S. W., Kim, D. S., Bai, D. H., & Yu, J. H. (2000). Applied Microbiology and Biotechnology, 54, 397–405.
Jae, J. G. (2005). Biotechnology Letters, 27, 1483–1486.
Sakka, K., Kusaka, R., Kawano, A., Karita, S., Sukhumavasi, J., Kimura, T., et al. (1998). Journal of Fermentation and Bioengineering, 86, 527–533.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Meruvu, H., Donthireddy, S.R.R. Purification and Characterization of an Antifungal Chitinase from Citrobacter freundii str. nov. haritD11. Appl Biochem Biotechnol 172, 196–205 (2014). https://doi.org/10.1007/s12010-013-0540-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12010-013-0540-4