Rational Engineering of a Thermostable Dipeptidase from <i>Thermobrachium celere</i> for Efficient Biosynthesis of <scp>l</scp> -Carnosine

l-Carnosine (β-alanyl-l-histidine) is a bioactive dipeptide widely used in pharmaceuticals, nutraceuticals, and functional foods. Conventional chemical synthesis of l-carnosine is energy-intensive and environmentally unfavorable, prompting interest in enzymatic alternatives. In this study, a thermostable dipeptidase from the thermophilic bacterium <i>Thermobrachium celere</i> (TcPepD) was identified and engineered for efficient l-carnosine synthesis. Wild-type TcPepD exhibited an optimal temperature of 70 °C and high thermal stability. Structure-guided mutagenesis based on AlphaFold modeling and molecular docking led to a double mutant, M2 (N117D/I169L), with a 2.7-fold increase in synthetic activity compared to the wild-type. Kinetic analysis showed that M2 displayed enhanced substrate affinity, particularly toward l-histidine, and improved catalytic efficiency. Molecular dynamics simulations revealed synergistic effects of the two mutations, resulting in improved substrate coordination and optimized active-site flexibility. Under high-substrate conditions at 65-70 °C, M2 produced up to 84.3 mM l-carnosine, demonstrating strong potential for high-temperature and high-efficiency biocatalytic production.