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

Abstract

The recombinant fructosyltransferase (Ftf) of Streptococcus salivarius was expressed in Escherichia coli and purified to electrophoretic homogeneity after a combination of adsorption, ion-exchange and gel-filtration chromatography. 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 activity 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 acceptors, including water, glucose and sucrose via a Ping Pong mechanism involving a fructosyl-enzyme intermediate. While this mechanism of catalysis is utilized by the levansucrases of Bacillus subtilis and Acetobacter diazotrophicus and the values 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 for the two other enzymes.

Mutation of aspartic acid residues in the fructosyltransferase of Streptococcus salivarius ATCC 25975

“…Parental and mutated proteins were expressed in E. coli NM522, extracted and purified as previously described [1]. At each stage of the purification, the specific polymer-forming activity of the Ftf proteins was quantified using [U-"%C]fructosyl-labelled sucrose [20] and the degree of purity monitored by SDS\PAGE [21].…”

“…The kinetic properties of the mutated Ftfs were determined as previously described following the detection of the amount of glucose and fructose formed at a given time with TC -Glucose\-Fructose kits supplied by Boehringer-Mannheim GmbH (Mannheim, Germany) [1]. The nature of the fructans produced in these reactions were analysed by thin layer chromatography [1].…”

“…The nature of the fructans produced in these reactions were analysed by thin layer chromatography [1].…”

“…The purified recombinant fructosyltransferase (Ftf) of Streptococcus sali arius catalyses the transfructosylation of sucrose by a Ping Pong mechanism involving a fructosyl-enzyme intermediate [1], in a manner similar to that of the levansucrases (sucrose : 2,6-β--fructan 6-β--fructosyltransferase ; EC 2.4.1.10) of Bacillus subtilis and Acetobacter diazotrophicus [2][3][4][5][6]. In an aqueous environment in which sucrose is the only substrate, two transfructosyl reactions initially occur resulting in the release of glucose.…”

“…water or a growing fructan chain in a sucrose solution), respectively. Comparison of the kinetic constants of the Ftf of S. sali arius with the levansucrases of B. subtilis and A. diazotrophicus suggests that the molecular basis of the first step of the catalytic pathway, the formation of a fructosyl-enzyme intermediate, EF, from sucrose, may have been conserved in these enzymes [1].…”