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Multistrategy Engineering of an Inulosucrase to Enhance the Activity and Thermostability for Efficient Production of Microbial Inulin.

Authors :
Ni D
Zhang S
Huang Z
Liu X
Xu W
Zhang W
Mu W
Source :
Journal of agricultural and food chemistry [J Agric Food Chem] 2024 Aug 14; Vol. 72 (32), pp. 18100-18109. Date of Electronic Publication: 2024 Aug 01.
Publication Year :
2024

Abstract

Inulin has found commercial applications in the pharmaceutical, nutraceutical, and food industries due to its beneficial health effects. The enzymatic biosynthesis of microbial inulin has garnered increasing attention. In this study, molecular modification was applied to Lactobacillus mulieris UMB7800 inulosucrase, an enzyme that specifically produces high-molecular weight inulin, to enhance its catalytic activity and thermostability. Among the 18 variable regions, R5 was identified as a crucial region significantly impacting enzymatic activity by replacing it with more conserved sequences. Site-directed mutagenesis combined with saturated mutagenesis revealed that the mutant A250 V increased activity by 68%. Additionally, after screening candidate mutants by rational design, four single-point mutants, S344D, H434P, E526D, and G531P, were shown to enhance thermostability. The final combinational mutant, M5, exhibited a 66% increase in activity and a 5-fold enhancement in half-life at 55 °C. These findings are significant for understanding the catalytic activity and thermostability of inulosucrase and are promising for the development of microbial inulin biosynthesis platforms.

Details

Language :
English
ISSN :
1520-5118
Volume :
72
Issue :
32
Database :
MEDLINE
Journal :
Journal of agricultural and food chemistry
Publication Type :
Academic Journal
Accession number :
39090787
Full Text :
https://doi.org/10.1021/acs.jafc.4c05224