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 , Lr LDH T247I/D249A/F306W/A214Y ( Lr LDH-M 1 ), had a viable catalytic advantage. It demonstrated a 3.3-fold increase in specific enzyme activity and approximately a 2.08-fold improvement of K cat . Correspondingly, molecular docking analysis provided a supporting explanation for the lower K m and higher K cat /K m 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 -M 1 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.