Enhanced Thermostability of an Improved l-Arabinose Isomerase Mutant for d-Tagatose Synthesis by Molecular Dynamics Simulations-Guided Rational Redesign of Flexible Regions
l-Arabinose isomerase (l-AI) catalyzes d-galactose to produce the rare, industrially important sugar d-tagatose. Enzyme stability is vital for its application in industrial processes, and rational design-based protein engineering methods have been employed to improve its stability. This study employed molecular dynamics simulations (MDS)-guided rational redesign of flexible regions to improve the thermostability of mesophilic Bifidobacterium adolescentis l-AI (Ba-l-AI). Comparative MDS (200 ns, 340–360 K) of thermophilic l-AIs (Geobacillus kaustophilus Gk-l-AI and Thermotoga maritima Tm-l-AI) and mesophilic Ba-l-AI identified flexible regions in Ba-l-AI via RMSF and ionic interaction. Five stabilizing mutations (G78C, K46R, N187R, K112R, and N190R) were introduced to provide rigidness. MDS of the mutant revealed reduced RMSF/RMSD, improved compactness, and enhanced conformational stability across temperatures, supported by free-energy landscape analysis resembling thermophilic profiles. Experimentally, the mutant exhibited superior thermal stability (72 °C), broader pH/temperature tolerance, and efficient d-tagatose production. The redesigned Ba-l-AI’s rigid backbone and stability highlight its potential for industrial d-tagatose biosynthesis.
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