In the determination of the activity of the free enzyme, the reaction medium consisted of acetate buffer (2.5 ml, 50 mM, pH 5.5), sucrose (0.1 ml, 300 mM).
Following a pre-incubation period (5 min at 35C), the assay was started by the addition of the enzyme solution (0.1 ml, 10 mg ml1) and incubation was continued for 5 min.
In order to terminate the enzymatic reaction, the reaction medium was then placed in a boiling water bath for 5 min.