Synthesis of ß-(2-6)-linked fructan with a partially purified levansucrase from Bacillus subtilis

Abstract

Levansucrase (EC 2.4.1.10) forms levan, a fructose homopolymer. The efficient production of fructans is affected by a few factors, including substrate concentration, temperature, pH, and the presence of enzyme cofactors. Conditions were thus optimized for the partially purified levansucrase from Bacillus subtilis DSM 347. The ratio of transferase to hydrolytic activity was determined by the LC–MS method. The synthesized fructan was analysed by two-dimension NMR spectra and GPC. The thermal stability at 50°C of the enzyme as well as the positive impact of Mn2+ ions at a concentration of 2.5mM are important properties of the practical use of the partially purified enzyme preparation. A significant change was recorded in the ratio of hydrolytic to transferase activity with Mn2+ ions and following preincubation of enzymes at the tested temperature range. Mn2+ ions at 2.5mM led to a 100% increase in transferase activity. Hydrolytic activity was four times higher with a low concentration (0.5mM) of Fe3+ ions. The maximum yield of fructan (using 83% of the introduced fructose) was obtained when the reaction ran at 50°C with the preincubated enzyme (1h at 50°C) at pH 5.5 in the presence of 2.5mM Mn2+.