ABSTRACT The chitin is extracted from the cuttlebone of S. inermis and the mineral contents are predictable. The structure and degree of deacetylation of extracted cuttlebone chitin is dogged through Fourier Transform Infrared (FT-IR) spectroscopy. The extracted cuttlebone chitin is used as the substrate for the production of chitinase from Vibrio sp. The extra cellular proteins are concentrated by ammonium sulphate precipitation, dialysed and then purified by using gel (sephadex G-100) chromatography. Among the 40 fractions, only two fractions (active fractions) showed maximum absorbance at 280 nm, which are pooled, dialysed and free-dried. The enzyme activity (0.104 μmoles/ml) and molecular weight (50 - 60 kDa) of purified chitinase is also determined through SDS- PAGE. The optimal condition for chitinase activity is pH between 6.0 - 6.5 and 45℃.
Cite this paper
Revathi, M. , Saravanan, R. and Shanmugam, A. (2012) Production and characterization of chitinase from Vibrio species, a head waste of shrimp Metapenaeus dobsonii (Miers, 1878) and chitin of Sepiella inermis Orbigny, 1848. Advances in Bioscience and Biotechnology, 3, 392-397. doi: 10.4236/abb.2012.34056.
 Svitil, L.A., Ni-Chandhain, M.S., Moore, A.J. and Kirchman, L.D. (1997) Chitin degradation proteins produced by the marine bacterium Vibrio harveyi growing on different forms of chitin. Applied Environmental Microbiology, 63, 408-413.
 Tombs, E. and Harding, A. (1998) In: An introduction to polysaccharide biotechnology, Taylor and Francis Publications, 144-151.
 Ravikumar, M.N.V. (2000) A review of chitin and chitosan applications. Reaction and Functional polymer, 46, 1-27.
 Dahiya, N., Tewari, R., Tiwari, P.R. and Hoondal, S.G. (2005) Chitinase from Enterobacter sp. NRG4: Its purification, characterization and reaction pattern. Electronic Journal of Biotechnology, 8(2), 134-145.
 Park, S.H., Lee, J. and Lee, W.C. (2000) Purification and characterization of chitinase from a marine bacterium Vibrio sp. 98CJ11027. Journal of Microbiology, 38, 224-229.
 Takiguchi, Y. (1991) Preparation of chiotosan and partially deacetylated chitin. In: Chitin, chitosanjikken manual, Otakara, A. and Yabuki, M. (Eds). Gihodou Shupan Kabushki Kasisha, Japan, pp 9-17.
 Tolaimate, A., Debrieres, J., Rhazi, M. and Alagui, A. (2003) Contribution to the preparation of chitins and chitosans with controlled physico–chemical properties. Polymers, 44,
 Shigemasa, Y., Matsuura, H., Sashwa, H. and Saimoto, H. (1996) Evaluation of different absorbance ratios from infrared spectroscopy for analyzing the degree of deacetylation in chitin. International Journal of Biological Macromolecule, 18, 237-242.
 Baxter, A., Dillon, M., Antony Taylor, K.D. and Roberts, G.A.F. (1992) Improved method for determination of the degree of N–acetylation of chitosan. International Journal of Biological Macromolecule, 14, 166-169.
 Wang, S.L., Lin, T.Y., Yen, Y.H., Liao, H.F. and Chen, Y.J. (2006) Bioconversion of shellfish chitin wastes for the production of Bacillus subtilis W-118 chitinase. Carbohydrate Research, 341, 2507-2515.
 Rodriguez-Kabana, R., Godoy, G., Morgan-Jones, G. and Shelby, R.A. (1983) Effects of chitin amendments to soil on Heterodera glycines, microbial populations and colonization of cysts by fungi. Plant Soil, 75, 95-106.
 Imoto, T. and Yagishita, K. (1971) A simple activity measurement of lysozyme. Agriculture Biological Chemistry, 35, 1154–1156.
 Imanaka, T., Fukui, T. and Fujiwara, S. (2001). Chitinase from Thermococcus kodakaraensis KOD1. Methods of Enzyme, 330, 319–329.
 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.
 Guo, S.H., Chen, J.K. and Lee, W.C. (2004) Purification and characterization of extracellular chitinase from Aeromonas schubertii. Enzyme Microbial Technology, 35, 550–556.
 Sambrook, J. and Russell, D.W. (2001) Molecular cloning: a laboratory manual, Cold Spring Habor Lab Press, pp A8.40-A8.51.
 Pearson, F.G., Marchessault, R.H. and Liang, C.Y. (1960) Infra red spectra of crystalline polysaccharides V chitin. Journal of Polymer Science, 13, 101-106.
 Baumano, P., Furaiss, A.L. and Lee, J.V. (1984) In: Bergey’s manual of systematic bacteriology, Krieg, N.R. and Holt, J.G. (Eds), Williams & Wilkins Co, London.
 Wang, S.L. and Chang, W.T. (1997) Purification and characterization of two bifunctional chitinases/lysozymes extracellularly produced by Pseudomonas aeruginosa K-187 in a shrimp and crab shell powder medium. Applied Environmental Microbiology, 63, 380-386.
 Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 277, 680-685.
 Patil, Y.T. and Satam, S.B. (2002) Chitin and chitosan. Treasure from. Crustacean shell waste. Sea Food Export Journal, 7, 31- 38.
 Zhou, N.S., Yang, Y.C., Huang, L., Cai, C.H. and Lin, Y.C. (1999) Isolation and characterization of chitinase from marine bacterium Vibrio sp. World Journal of Microbiology and Biotechnology, 15, 745-746.