PURIFICATION AND CHARACTERIZATION OF BETA-FRUCTOFURANOSIDASE (INVERTASE) FROM AN ORAL-STRAIN OF STREPTOCOCCUS-MITIS

β-Fructofuranosidase (EC 3.2.1.26) isolated from the cell extract of Streptococcus mitis ATCC 903 was purified 167-fold. The enzyme appeared homogeneous by gel electrophoresis. In gel electrofocusing, two protein bands, identical with two bands of β-fructofuranosidase activity, were visualized at pI values 4.05 and 4.25. The mol. wt of the enzyme was estimated to be 49,000 and the Km for sucrose was 7.4 × 10-2M. The enzyme had optimal activity in the pH-range 6.3-7.2. The sulphhydryl reagents 2-mercaptoethanol, p-chloromercuribenzoate, N-ethylmaleimide and iodoacetate and the protein denaturants urea and sodium dodecylsulphate were inhibitory. EDTA and sodium fluoride had no effect on the activity. Inorganic phosphate had a stimulatory effect on enzyme activity. Apparent Km for inorganic phosphate was 2.1 × 10-2M. The enzyme obeyed Michaelis-Menten kinetics. Variation in pH did not change the shapes of the saturation curves for sucrose. The Hill coefficient was calculated to be 1.0. No stimulatory or inhibitory effect of glycolytic intermediates and nucleotides was found. These results indicate that β-fructofuranosidase is a non-allosteric enzyme. © 1979.