Physiologia plantarum vol:98 issue:3 pages:455-466
A genuine 1-SST (sucrose:sucrose 1-fructosyl transferase, EC 18.104.22.168) was purified and characterized from young chicory roots (Cichorium intybus L. var. foliosum cv. Flash) by a combination of ammonium sulfate precipitation, concanavalin A affinity chromatography, anion and cation exchange chromatography. This protocol produced a 63-fold purification and a specific activity of 4.75 U (mg protein)(-1). The mass of the enzyme was 69 kDa as estimated by gel filtration. On SDS-PAGE apparent molecular masses of 49 kDa (alpha-subunit) and 24 kDa (beta-subunit) were found. Further specification was obtained by MALDI-TOF MS detecting molecular ions at m/z 40 109 and 19 896. These two fragments were also found on a western blot using an SDS-boiled chicory root extract and chicken-raised polyclonal antibodies against the purified 1-SST, indicating that the enzyme is a heterodimer in vivo. The N-terminus of chicory root 1-SST alpha-subunit was shown to be highly homologous with the cDNA-derived amino acid sequences from barley 6-SFT and a number of beta-fructosyl hydrolases (invertases and fructan hydrolases). However, chicory root 1-SST properties could be clearly differentiated from those of chicory root 1-FFT (EC 22.214.171.124), chicory root acid invertase (EC 126.96.36.199) and yeast invertase. The enzyme mainly produced 1-kestose and glucose from physiologically relevant sucrose concentrations, indicating that this 1-SST is the key enzyme initiating fructan biosynthesis in vivo. However, like chicory root 1-FFT and barley 6-SFT, the enzyme also showed some beta-fructofuranosidase activity (fructosyl transfer to water) at very low sucrose concentrations. Although sucrose clearly is the best substrate for the enzyme, some transferase and beta-fructofuranosidase activity were also detected using 1-kestose as the sole substrate.