Computationally Informed Redesign of Levansucrase from <i>Erwinia tasmaniensis</i> to Enhance Its Thermostability for Levan Biosynthesis

Levan is a versatile biomaterial because of its unique physicochemical properties and bioactivities. To synthesize levan efficiently, it is important to improve the thermostability of levansucrase. This study presents the first engineering study on <i>Erwinia tasmaniensis</i> levansucrase (<i>Et</i>Lsc) employing a rational protein design approach. Molecular dynamics (MD) simulations were used to identify thermally sensitive regions of <i>Et</i>Lsc, and thermostable variants were designed by using FireProt folding energy calculations. Among the designed candidates, the A197P and S239P mutants had largely higher melting temperatures (<i>T</i>_m) and half-life (<i>t</i>_1/2) compared to the wild type. The double variant A197P/S239P exhibited a 7.9 °C increase in <i>T</i>_m and a 48-fold extension of <i>t</i>_1/2 at 50 °C, which represents a more significant enhancement than previous studies. Kinetic and product analyses using HPSEC, HPAEC-PAD, and ¹H NMR demonstrated that these mutations did not alter the catalytic efficiency or levan structure. The results demonstrate the potential of MD-aided energy-based engineering for thermostable <i>Et</i>Lsc designs.