Abstract:β-(2,6) levansucrase (LS) with high thermostability was identified and the efficient synthesis of levan by levansucrase was studied. High-temperature molecular dynamics simulation was used to screen the LS with potential high thermostability. The target enzyme was heterogeneously expressed in Escherichia coli BL21(DE3) and purified through one-step Ni2+-affinity purification. The thermostability of LS was determined by incubating the enzyme at different temperatures, and the highest conversion was obtained through the optimization of conditions. The Cedi-LS showed the highest activity at 65 ℃, which was much higher than the other identified LSs. Meanwhile, Cedi-LS remained more than 90% active when incubated at 45 ℃ for 72 h, and it also remained more than 60% of its initial activity at 55 ℃ for 60 h, exhibiting excellent thermostability. The Cedi-LS produced oligosaccharides, low molecular weight (LMW) β-(2,6) levan, and high molecular weight (HMW) β-(2,6) levan simultaneously during reaction. When the temperature decreased from 65 to 35 ℃, Cedi-LS tended to produce HMW-levan with a Mw of 8.4×106 Da. At pH 5.5 and 35 ℃, the equilibrium conversion ratio from sucrose to levan was 41.4% with 30% sucrose and 20 μg/mL enzyme. In summary, an extreme thermostable LS was screened which could efficiently produce levan with different molecular weights.
