Computational design of Bacillus licheniformis RN-01 levansucrase for control of the chain length of levan-type fructooligosaccharides.

Levansucrase (LS) from Gram-positive bacteria generally produces a large quantity of levan polymer, a polyfructose with glucose at the end (GFn) but a small quantity of levan-type fructooligosaccharides (LFOs). The properties of levan and LFOs depend on their chain lengths, thereby determining their potential applications in food and pharmaceutical industries such as prebiotics and anti-tumor agents. Therefore, an ability to redesign and engineer the active site of levansucrase for synthesis of products with desired degree of polymerization (DP) is very beneficial. We employed computational protein design, docking and molecular dynamics to redesign and engineer the active site of Bacillus licheniformis RN-01 levansucrase for production of LFOs with DP up to five (GF4), using two approaches: 1) blocking oligosaccharide binding track of GF3-LS complex with large aromatic residues and 2) eliminating hydrogen bond interactions between terminal glucose of GF4 and side chains of binding residues of GF4-LS complex. The designed enzymes and their product patterns from these two approaches were experimentally characterized. The experimental results show that the first approach was successful in creating N251W and N251W/K372Y mutants that synthesized LFOs with DP up to five. This work illustrates how computer-aided approaches can offer novel opportunities to engineer enzymes for desired products.