Purification and characterization of chitinase from the integument of the Mediterranean flour moth, Ephestia kuehniella (Lepidoptera: Pyralidae) and its antibacterial role
Chitinases are found in species from all kingdoms, including Kingdom Animalia. We purified chitinase, poly [1,4-(N-acetyl-β-D-glucosaminide)] glycanohydrolase, by cation exchange chromatography on SP-Sepharose from the integument of a lepidopteran, the Mediterranean flour moth, Ephestia kuehniella Zeller, and further characterized it. Ephestia kuehniella chitinase has similar properties to other insect chitinases, with respect to the molecular mass. Its molecular mass was 79.7 kDa by sodium dodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature for activity were 6 and 40 °C, respectively. The purified enzyme exhibited stability at a pH range from 4.5 to 8.0 and showed high sensitivity to Co2+, Mn2+, Ca2+, Hg2+ and Ag+. The Km and Vmax values of E. kuehniella chitinase were, respectively, 3.98 mg/mL and 7.11 μmol/min/mg for colloidal chitin. In comparison to the values determined for the substrate affinity, the enzyme had a 2.36-fold higher affinity for Carboxymethyl-Chitin-Remazol Brilliant Violet (CM-Chitin-RBV) as a soluble substrate. We examined the antibacterial effect of E. kuehniella chitinase on the growth of both Gram-positive and Gram-negative bacteria. The enzyme inhibited the growth of Bacillus subtilis, Bacillus thuringiensis and Escherichia coli, where the average diameter of inhibition zone was 20, 23 and 19 mm, respectively. The antimicrobial effect and chitinase activity strengthen the hypothesis that the enzyme could be used in biological control against plant pests.
Key wordsEphestia kuehniella insect chitinase integument chitin SP-Sepharose antibacterial effect
Unable to display preview. Download preview PDF.
- Ehrlich H, Maldonado M., Spindler K.-d., Eckert C., Hanke T, Born R., Goebel C., Simon P., Heinemann S. and Worch H. (2007b) First evidence of chitin as a component of the skeletal fibers of marine sponges. Part I. Verongidae (Demospongia: Porifera). Journal of Experimental Zoology Part B 308, 347–356.CrossRefGoogle Scholar
- Jagadeeswari S. J. and Panneer Selvam K. (2012) Optimization of chitinase production by soil Streptomyces sp. SJKP9. Journal of Academia and Industrial Research 1, 332–336.Google Scholar
- Mathur A., Rawat A., Bhatt G., Baweja S., Ahmad F., Grover A., Madhav K., Dhand M., Mathur D., Verma S. K., Singh S. K. and Dua V. K. (2011) Isolation of Bacillus producing chitinase from soil: Production and purification of chito-oligosaccharides from chitin extracted from fresh water crustaceans and antimicrobial activity of chitinase. Recent Research in Science and Technology 3(11), 01–06.Google Scholar