Purification and biochemical characterization of a pathogenesis related endochitinase Arachis hypogaea

Abstract

Plants protect themselves by producing phytoalexins and pathogenesis related (PR) proteins in response to different physical and biological stresses as well as against pathological attack. In the present study, chitinase, a PR protein, from peanut seeds was purified to homogeneity by ammonium sulfate precipitation, gel filtration chromatography on Sephadex G-75 column and ion exchange chromatography on Q-Sepharose column. Chitinase was identified as monomeric protein with molecular mass of 30 kDa by SDS-PAGE. Chitinase activity was also evaluated on chitin agar plates showing distinct zones. The optimum pH and temperature of purified chitinase activity were found to be 5.2 and 35°C, respectively. The enzyme was stable within broad pH range of 3.6 to 7.6 and up to 55°C temperature. Amongst the various substrates, acid swollen chitin was found to be the best substrate for chitinase when used at the concentration of 1% exhibiting its high specificity in catalyzing glycosidic bonds between N-acetylglucosamine residues. Chitinase activity of the purified enzyme was inhibited by Hg2+ and Ag2+ at 1 mM concentration and enhanced by Ca2+, Mg2+. The increase in the presence of 2-mercaptomethanol demonstrates the existence of sulfhydryl groups in enzyme active site. The enzyme exhibited a strong inhibitory action towards a variety of fungal species including Fusarium oxysporum, Trichoderma reesei, Aspergillus flavus and A. niger. Purified chitinase showed fungicidal properties against A. flavus on white bread and therefore, could be applied in bakery products to inhibit the formation and growth of fungal colonies.