Design of chimeric levansucrases with improved transglycosylation activity

Fructansucrases (FSs), including levansucrases and inulosucrases, are enzymes that synthesize fructose polymers from sucrose by the direct transfer of the fructosyl moiety to a growing polymer chain. These enzymes, particularly the single domain fructansucrases, also possess an important hydrolytic activity, which may account for as much as 70 to 80% of substrate conversion, depending on reaction conditions. Here, we report the construction of four chimeric levansucrases from SacB, a single domain levansucrase produced by Bacillus subtilis. Based on observations derived from the effect of domain deletion in both multidomain fructansucrases and glucansucrases, we attached different extensions to SacB. These extensions included the transitional domain and complete C-terminal domain of Leuconostoc citreum inulosucrase (IslA), Leuconostoc mesenteroides levansucrase (LevC), and a L. mesenteroides glucansucrase (DsrP). It was found that in some cases the hydrolytic activity was reduced to less than 10% of substrate conversion; however, all of the constructs were as stable as SacB. This shift in enzyme specificity was observed even when the SacB catalytic domain was extended only with the transitional region found in multidomain FSs. Specific kinetic analysis revealed that this change in specificity of the SacB chimeric constructs was derived from a 5-fold increase in the transfructosylation k(cat) and not from a reduction of the hydrolytic k(cat), which remained constant.