Purification and characterization of tomatinase from Fusarium oxysporum f sp lycopersici

The antifungal compound alpha-tomatine, present in tomato plants, has been reported to provide a preformed chemical barrier against phytopathogenic fungi, Fusarium oxysporum f. sp, lycopersici, a tomato pathogen, produces an extracellular enzyme inducible by or-tomatine. This enzyme, known as tomatinase, catalyzes the hydrolysis of alpha-tomatine into its nonfungitoxic forms, tomatidine and beta-lycotetraose. The maximal tomatinase activity in the fungal culture medium was observed after 48 h of incubation of germinated conidia at an alpha-tomatine concentration of 20 mu g/ml. The enzymatic activity in the supernatant was concentrated against polyethylene glycol 35000, and the enzyme was then purified to electrophoretic homogeneity bg a procedure that includes preparative isoelectric focusing and preparative gel electrophoresis as main steps, The purification procedure had a yield of 18%, and the protein was purified about 40-fold, Tomatinase was found to be a monomer of 50 kDa by both native gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, The analytical isoelectric focusing of the native tomatinase showed at least five isoforms with pIs ranging from 4.8 to 5.8, Treatment with N-glycosidase F gave a single protein band of 45 kDa, indicating that the 50-kDa protein was N glycosylated. Tomatinase activity was optimum at 45 to 50 degrees C and at pH 5.5 to 7, The enzyme was stable at acidic pH and temperatures below 50 degrees C. The enzyme had no apparent requirement for cofactors, although Co2+ and Mn2+ produced a slight stimulating effect on tomatinase activity. Kinetic experiments at 30 degrees C gave a K-m of 1.1 mM for alpha-tomatine and a V-max of 118 mu mol/min/mg. An activation energy of 88 kJ/mol was calculated.