Levansucrase (EC 2.1.4.10, LS) has been used in the production of levan and levan-type fructooligosaccharides from sucrose; however, development of further application is restricted due to its poor thermostability. The LS from Brenneria sp. EniD312 was engineered using a structure-guided approach. Residue Glu404 was located in the "-TEAP-" motif and varied among LSs with different thermostabilities. Site-directed mutagenesis was performed in Glu404 and thermostability was evaluated by measuring the half-life and structural melting temperature (Tm) of the wild-type LS and its Glu404-mutant variants. The optimal temperature for the Glu404 mutants was similar to that of the wild-type enzyme, however, the Tm of E404 L mutant was enhanced by 2.8 °C and the half-life was increased by 12.5- and 1.3- fold at 35 and 45 °C, respectively. The other mutants E404 W, E404 V, E404I, and E404 F also showed a pronounced increase in Tm and thermostability. Finally, the improvement of thermostability of LS through mutation in Glu404 belonging to the "-TEAP"- motif could be ascribed to the change of microenvironment in the LS structure. The change of the micro-environment mainly included the enhanced structural stability between two β-hairpins and the elevated hydrophobic interactions in the overall protein structure. This work proposes new insights into the thermostabilization mechanism of other LSs.
Keywords: Glu(404); Levan; Levansucrase; Site-directed mutagenesis; Thermostability.
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