Induction, Purification and Characterization of Chitinases from Chickpea ( Cicer Arietinum L.) Leaves and Pods Infected with Ascochyta rabiei

Abstract

Chitinase (EC 3.2.1.14) activity increased in leaves and pods of resistant as well as susceptible cultivars of chickpea ( Cicer arietinum L.) inoculated with Ascochyta rabiei . Leaves of resistant cultivars exhibited an about five-fold increase on the 6th day of inoculation, as compared with the uninoculated control. Although a similar pattern of changes in enzyme activity was discerned with the susceptible cultivar also, the magnitude of induction was low. Compared with leaves, the excised inoculated pods showed a rapid and early increase in chitinase activity. Chitinase was purified from leaves and pods of inoculated chickpea resistant cultivar 30- and 33-fold, respectively, by heat treatment, ammonium sulfate fractionation, gel filtration and affinity chromatography on regenerated chitin. Except for the molecular weight (leaf, 30 kD; pod 27 kD), the enzyme from both sources had the same properties with an optimum pH of 5.0, optimum temperature of 37 °C and a Km value of 0.65 mg chitin mL −1 . The enzyme had no requirement for any of the metal ions, and rather was inhibited by He 2+ .