Enhanced catalytic activity and stability of lactate dehydrogenase for cascade catalysis of D-PLA by rational design.

Serving as a vital medical intermediate and an environmentally-friendly preservative, D-PLA exhibits substantial potential across various industries. In this report, the urgent need for efficient production motivated us to achieve the rational design of lactate dehydrogenase and enhance catalytic efficiency. Surprisingly, the enzymatic properties revealed that a mutant enzyme, LrLDHT247I/D249A/F306W/A214Y (LrLDH-M1), had a viable catalytic advantage. It demonstrated a 3.3-fold increase in specific enzyme activity and approximately a 2.08-fold improvement of Kcat. Correspondingly, molecular docking analysis provided a supporting explanation for the lower Km and higher Kcat/Km of the mutant enzyme. Thermostability analysis exhibited increased half-lives and the deactivation rate constants decreased at different temperatures (1.47-2.26-fold). In addition, the mutant showed excellent resistance abilities in harsh environments, particularly under acidic conditions. Then, a two-bacterium (E. coli/pET28a-lrldh-M1 and E. coli/pET28a-ladd) coupled catalytic system was developed and realized a significant conversion rate (77.7%) of D-phenyllactic acid, using 10 g/L L-phenylalanine as the substrate in a two-step cascade reaction.