Rational Engineering of a Thermostable Dipeptidase from Thermobrachium celere for Efficient Biosynthesis of l -Carnosine

(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.