Enzymatic fructosylation of aromatic and aliphatic alcohols by Bacillus subtilis levansucrase: Reactivity of acceptors

Levansucrases from Bacillus subtilis (BS-LVS) and Leuconostoc mesenteroides ssp. mesenteroides ATCC 8293 (LevC), inulosucrase from Leuconostoc citreum (IslA) and an invertase from Saccharomyces cerevisiae (Inv) were evaluated in acceptor reactions with non-sugar acceptors. Among them, BS-LVS was selected for the fructosylation of aromatic or aliphatic alcohols due to its high activity and stability. The effects of acceptor concentration, enzyme concentration and the presence of a co-solvent in the fructosylation efficiency of hydroquinone were evaluated. It was demonstrated that this reaction is kinetically controlled, producing the best yields of phenolic fructosides when 500 mM of acceptor and 5 U mL−1 of enzyme were employed. Higher enzyme loads resulted in the rapid hydrolysis of the products. Increased amounts of organic co-solvent up to 50% (v/v) reduced fructoside yield due to a concomitant decrease in the thermodynamic activity of the acceptor, as confirmed by theoretical calculations using COSMO-RS; moreover, increased fructose transfer to levan and reduced hydrolysis were observed. It was found that BS-LVS preferentially fructosylates aromatic over aliphatic alcohols. A maximum fructoside production (19–35 mM) was obtained with dihydroxybenzene acceptors such as hydroquinone, whereas reactions with primary alcohols, such as benzyl alcohol resulted in lower fructosylation efficiency. This selectivity was also demonstrated by the fact that 4-hydroxybenzylalcohol, a bifunctional acceptor, was fructosylated at a rate ten times faster on its aromatic hydroxyl group. BS-LVS selectivity over phenol fructosylation was inversely correlated with the acceptor pKa value.

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► Novel phenolic fructosides were synthesized by B. subtilis levansucrase.
► This reaction is influenced by structure and thermodynamic activity of the acceptors.
► The highest fructoside yields were obtained with dihydroxybenzene acceptors.
► Fructosylation yield was inversely related to acceptor pKa.