Enhancement of the Brenneria sp. levansucrase thermostability by site-directed mutagenesis at Glu404 located at the -TEAP- residue motif

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 Glu(404) was located in the -TEAP- motif and varied among LSs with different thermostabilities. Site-directed mutagenesis was performed in Glu(404) and thermostability was evaluated by measuring the half-life and structural melting temperature (T-m) of the wild-type LS and its Glu(404)-mutant variants. The optimal temperature for the Glu(404) mutants was similar to that of the wild-type enzyme, however, the T-m of E404 L mutant was enhanced by 2.8 degrees C and the half-life was increased by 12.5- and 1.3- fold at 35 and 45 degrees C, respectively. The other mutants E404 W, E404 V, E4041, and E404 F also showed a pronounced increase in T-m and thermostability. Finally, the improvement of thermostability of LS through mutation in Glu(404) 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 beta-hairpins and the elevated hydrophobic interactions in the overall protein structure. This work proposes new insights into the thermostabilization mechanism of other LSs.