K. Lairini et al., PURIFICATION AND CHARACTERIZATION OF TOMATINASE FROM FUSARIUM-OXYSPORUM F-SP LYCOPERSICI, Applied and environmental microbiology, 62(5), 1996, pp. 1604-1609
The antifungal compound alpha-tomatine, present in tomato plants, has
been reported to provide a preformed chemical barrier against phytopat
hogenic fungi, Fusarium oxysporum f. sp, lycopersici, a tomato pathoge
n, produces an extracellular enzyme inducible by or-tomatine. This enz
yme, known as tomatinase, catalyzes the hydrolysis of alpha-tomatine i
nto its nonfungitoxic forms, tomatidine and beta-lycotetraose. The max
imal tomatinase activity in the fungal culture medium was observed aft
er 48 h of incubation of germinated conidia at an alpha-tomatine conce
ntration of 20 mu g/ml. The enzymatic activity in the supernatant was
concentrated against polyethylene glycol 35000, and the enzyme was the
n 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 monom
er of 50 kDa by both native gel electrophoresis and sodium dodecyl sul
fate-polyacrylamide gel electrophoresis, The analytical isoelectric fo
cusing 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 glyc
osylated. 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 belo
w 50 degrees C. The enzyme had no apparent requirement for cofactors,
although Co2+ and Mn2+ produced a slight stimulating effect on tomatin
ase 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 en
ergy of 88 kJ/mol was calculated.