Purification, Characterization Of A Chitinase From Peanut (Arachis Hypogaea)

A chitinase with antifungal activity was isolated from peanut (Arachis hypogaea) seeds. The procedure entailed aqueous extraction, affinity chromatography on Affi-gel, ion-exchange chromatography on CM-Sepharose and Gel- filtration chromatography on Sephadex G-75. The enzymatic activity of pure chitinase is 46 times higher than the crude extracts. This chitinase showed a single band on SDS-PAGE with a molecular weight of 34.4 k Da both in reduced and oxidized conditions, indicating it is a monomeric protein. By using isoelectric focusing electrophoresis (IEF), the isoelectric point (pI) was determined as 5.1. The acidic isoelectric point showed the chitinase is an acidic enzyme.The enzyme activity of the chitinase is determined by the method of DNS. The optimum pH was 5.4 and the optimum temperature was 40-50℃. The purified enzyme lost enzymatic activity at pH 6.0 and also at temperature below 40 ℃ or above50℃. The enzyme was stable below 50℃, but was rapidly inactivated when incubatedat temperatures above 55℃. It was completely inactivated after incubation at atemperature above 75 ℃.The antioxidant activity of the chitinase was determined by DPPH assay. Thescavenging activity can attain 63.5% when the concentration was 180 μg /ml. And theresult indicated that it has good scavenging effect on DPPH.It exhibited antifungal activity against the fungi Physalospora piricola, Fusariumoxysporum, Pythium apHanidermatum, Botrytis cinerea and Alternaria alternata (Fr.)Keissl by the disk agar diffusion and oxford cup method. The IC50 value of theantifungal activity of the chitinase was carried out toward Botrytis cinerea. The IC50was calculated to be 18.9μM.The chitinase displayed inhibition activity to the cell of HepG 2 by the method ofMTT, with IC50 252μg/ml. Compared with the IC50 value of MDCK 296μg/ml, it...