# Purification and characterization of a thermophilic NAD +‐dependent lactate dehydrogenase from Moorella thermoacetica

## TL;DR

This paper studies a key enzyme in lactate metabolism from a thermophilic bacterium, showing its potential for biotechnology.

## Contribution

The purification and characterization of a NAD+‐dependent lactate dehydrogenase from Moorella thermoacetica is presented.

## Key findings

- The enzyme shows high activity for lactate oxidation at 65 °C and pH 8.
- NADH-driven pyruvate reduction is significantly faster than lactate oxidation.
- The enzyme favors lactate formation, suggesting potential for biotechnological improvements.

## Abstract

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.

The thermophilic acetogenic model organism Moorella thermoacetica can utilize lactate as substrate. The formation of lactate by reduction of pyruvate is energetically favorable, but the reverse reaction, the oxidation of lactate to pyruvate with NAD+ as electron acceptor is challenging. To shed light into enzymology of the lactate metabolism in M. thermoacetica we purified and characterized the NAD+‐dependent l‐lactate dehydrogenase.