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The antifungal compound ␣-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 ␣-tomatine. This enzyme, known as tomatinase, catalyzes the hydrolysis of ␣-tomatine into its nonfungitoxic forms, tomatidine and ␤-lycotetraose. The maximal tomatinase activity in the fungal culture medium was observed after 48 h of incubation of germinated conidia at an ␣-tomatine concentration of 20 g/ml. The enzymatic activity in the supernatant was concentrated against polyethylene glycol 35000, and the enzyme was then purified to electrophoretic homogeneity by 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؇C and at pH 5.5 to 7. The enzyme was stable at acidic pH and temperatures below 50؇C. The enzyme had no apparent requirement for cofactors, although Co 2؉ and Mn 2؉ produced a slight stimulating effect on tomatinase activity. Kinetic experiments at 30؇C gave a K m of 1.1 mM for ␣-tomatine and a V max of 118 mol/min/mg. An activation energy of 88 kJ/mol was calculated.

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