title: Purification and characterization of tomatinase from Fusarium oxysporum f. sp. lycopersici.
abstract: 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 alpha-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 micrograms/ml. The enzymatic activity in the supernatant was concentrated against polyethylene glycol 35,000, 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 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 Vmax of 118 mumol/min/mg. An activation energy of 88 kJ/mol was calculated.
authors.affiliation: Departamento de Genética, Facultad de Ciencias, Universidad de Córdoba, Spain.
authors.forename: K
authors.initials: K
authors.lastname: Lairini
authors.forename: A
authors.initials: A
authors.lastname: Perez-Espinosa
authors.forename: M
authors.initials: M
authors.lastname: Pineda
authors.forename: M
authors.initials: M
authors.lastname: Ruiz-Rubio
mesh_terms: D004798:Enzymes / Q000302:isolation & purification* / Q000378:metabolism; D005670:Fusarium / Q000201:enzymology*; D006026:Glycoside Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D014053:Tomatine / Q000378:metabolism
chemical_list: D004798:Enzymes; D014053:Tomatine; D006026:Glycoside Hydrolases; C099475:tomatinase