A chitinase from Adenanthera pavonina L. seeds: purification, characterisation and immunolocalisation

Abstract

Chitinases are hydrolases involved in plant defense against a variety of pathogens, including fungi. The antifungic activities of these proteins consist mainly of their capacity to act on chitin, one of the most abundant components of the fungal cell wall. Chitinases have been isolated from various species and organs of plants, including seeds. The objective of this work was the purification, characterisation and immunolocalisation of a thermostable chitinase from seeds of Adenanthera pavonina L. Flour from seeds was initially extracted for 2 h at 4 °C with extraction buffer (10 mM Na 2 HPO 4 , 15 mM NaH 2 PO 4 , 100 mM KCl, 1.5% EDTA) in the proportion of 1:5 (m:v) and then submitted to ammonium sulphate fractionation. Chitinase activity was detected in the fraction between 30 and 70% relative ammonium sulphate saturation. This fraction was submitted to different chromatographic methods for the purification of the enzyme: Sephacryl S-200, Phenyl-Sepharose and chitin affinity. Chitinase activity was assayed using the substrate fluorescent 4-metilumbeliferona-β- d - N , N ′, N ″-triacetilquitotriose and monitored by SDS-PAGE chitinase electrophoresis. The purified enzyme presented a molecular mass of approximately 30 kDa, and a higher at pH 4.0. The optimal activity temperature for the enzyme was 60 °C, however, activity was maintained above 70 °C. The tissue and subcellular localisation of the enzyme indicated that isolated chitinase was mainly localised in the cytosolic compartments. In addition, the presence of a similar protein to the isolated chitinase was detected in exudates from seeds, discharged during germination.