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Purification and characterization of chitinase from the integument of the Mediterranean flour moth, Ephestia kuehniella (Lepidoptera: Pyralidae) and its antibacterial role

Published online by Cambridge University Press:  17 January 2018

Mahmoud Mehranian ,
Reza Farshbaf Pourabad ,
Nemat Sokhandan-Bashir ,
Ahmad Asoodeh  and
Ahsan Salihi
Mahmoud Mehranian*
Affiliation:
Department of Plant Protection, Faculty of Agriculture, University of Tabriz, Tabriz, 51666-14888, Iran
Reza Farshbaf Pourabad
Affiliation:
Department of Plant Protection, Faculty of Agriculture, University of Tabriz, Tabriz, 51666-14888, Iran
Nemat Sokhandan-Bashir
Affiliation:
Department of Plant Protection, Faculty of Agriculture, University of Tabriz, Tabriz, 51666-14888, Iran
Ahmad Asoodeh
Affiliation:
Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran
Ahsan Salihi
Affiliation:
Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran
*
*E-mail: [email protected]
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Abstract

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.

Keywords

Ephestia kuehniellainsect chitinaseintegumentchitinSP-Sepharoseantibacterial effect
Type
Research Paper
Information
International Journal of Tropical Insect Science , Volume 38 , Issue 2 , June 2018 , pp. 105 - 116
Copyright
Copyright © icipe 2018 

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