Enhancement of the Brenneria sp. levansucrase thermostability by site-directed mutagenesis at Glu 404 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. R...
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Published in | Journal of biotechnology Vol. 290; p. 1 |
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Main Authors | , , , , , |
Format | Journal Article |
Language | English |
Published |
Netherlands
29.11.2018
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Subjects | |
Online Access | Get full text |
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Summary: | 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
was located in the "-TEAP-" motif and varied among LSs with different thermostabilities. Site-directed mutagenesis was performed in Glu
and thermostability was evaluated by measuring the half-life and structural melting temperature (T
) of the wild-type LS and its Glu
-mutant variants. The optimal temperature for the Glu
mutants was similar to that of the wild-type enzyme, however, the T
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 T
and thermostability. Finally, the improvement of thermostability of LS through mutation in Glu
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. |
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ISSN: | 1873-4863 |