Induction of proteases secreted by fusarium oxysporum F. SP. Dianthi in the presence of carnation root cell walls. Biochemical characterization of a serine protease

Abstract

We determined that protease activity is secreted by Fusarium oxysporum f. sp. dianthi race 2 (Fod) in vitro. The cell wall extracted from resistant and susceptible cultivars of carnation (Dianthus caryophyllus L.) was used as an inducer. The activity increased in the presence of the two cultivars, and higher levels were detected with the susceptible cultivar. One of the three proteases secreted by Fod was purified using ion exchange (DEAE-cellulose), gel filtration (Sephadex G-75), and affinity chromatographies (Benzamidine Sepharose 6B). Using protease inhibitors, the purified enzyme was classified as a trypsin-like serine protease that was inhibited by phenylmethanesulfonyl fluoride, diisopropylfluorophosphate and the soybean trypsin inhibitor. In addition, the enzyme was biochemically characterized and determined to have an optimal pH of 8.0, an optimal temperature of 50°C, stability from pH 6 to 8, temperature stability between 40 to 60°C, a molecular weight of 54 kDa, and an isoelectric point of 4.4. The values of the Km and Vmax kinetic constants were found to be 0.31 mg/ml and 24.7 µmol/min, respectively.