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

Publication date: Available online 29 November 2018Source: Journal of BiotechnologyAuthor(s): Wei Xu, Jiaying Peng, Wenli Zhang, Tao Zhang, Cuie Guang, Wanmeng MuAbstractLevansucrase (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 protei...
Source: Journal of Biotechnology - Category: Biotechnology Source Type: research