Insights into the heterogeneity of levan polymers synthesized by levansucrase Bs-SacB from Bacillus subtilis 168

Levan is an enzymatically synthesized fructose polymer with widely reported structural heterogeneity depending on the producing levansucrase, the reaction conditions employed for its synthesis and the characterization techniques. We studied here the specific properties of levan produced by recombinant levansucrase from B. subtilis 168 (Bs-SacB), often characterized as a bimodal distribution, that is, a mixture of low and high molecular weight levan. We found significant differences between both levans in terms of the already reported molecular weight, size and morphology using different analytical methods. The low molecular weight levan consists of a non-uniform polymer ranging from 50 to 230 kDa, synthesized through a non-processive mechanism that can spontaneously form spherical nanoparticles in the reaction medium. In contrast, high molecular weight levan is a uniform polymer, most probably synthesized through a processive mechanism, with an average molecular weight of 30,750 kDa and a poorly defined nano-structure. This is the first report exploring differences in morphology between low and high molecular weight levans. Our findings demonstrate that only the low molecular weight levan forms spherical nanoparticles in the reaction medium and that high molecular weight levan is mainly composed of a 33,000 kDa fraction with a microgel behavior.