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.