Purification and enzymic properties of the fructosyltransferase of Streptococcus salivarius ATCC 25975

The recombinant fructosyltransferase (Ftf) of Streptococcus salivarius was expressed in Escherichia coli and purified to electrophoretic homogeneity a fter a combination of adsorption, ion-exchange and gel-filtration chromatog raphy. The N-terminal signal sequence of the Ftf was removed by E. coli at the same site as in its natural host. The purified Ftf exhibited maximum ac tivity at pH 6.0 and 37 degrees C, was activated by Ca2+, but inhibited by the metal ions Cu2+, Zn2+, Hg2+ and Fe3+. The enzyme catalysed the transfer of the fructosyl moiety of sucrose to a number of accepters, including wat er, glucose and sucrose via a Ping Pong mechanism involving a fructosyl-enz yme intermediate. While this mechanism of catalysis is utilized by the leva nsucrases of Bacillus subtilis and Acetobacter diazotrophicus and the value s of the kinetic constants for the three enzymes are similar, sucrose was a far more efficient fructosyl-acceptor for the Ftf of S. salivarius than fo r the two other enzymes.