Purification and characterization of a thermophilic NAD+-dependent lactate dehydrogenase from Moorella thermoacetica.

Oxidation of lactate under anaerobic dark fermentative conditions poses an energetic problem. The redox potential of the lactate/pyruvate couple is too electropositive to reduce the physiological electron carriers NAD(P)⁺ or ferredoxin. However, the thermophilic, anaerobic, and acetogenic model organism Moorella thermoacetica can grow on lactate but was suggested to have a NAD⁺-dependent lactate dehydrogenase (LDH), based on enzyme assays in cell-free extract. LDHs of thermophilic and anaerobic bacteria are barely characterized but have a huge biotechnological potential. Here, we have purified the LDH from M. thermoacetica by classical chromatography. Lactate-dependent NAD⁺ reduction was observed with high rates. Electron bifurcation was not observed. At pH 8 and 65 °C, the LDH had a specific activity of 60 U·mg^-1 for lactate oxidation, but NADH-driven pyruvate reduction was around four times faster with an activity of 237 U·mg^-1. Since lactate formation is preferred by the enzyme, further modifications of the LDH can be suggested to improve the kinetics of this enzyme making it a promising candidate for biotechnological applications.