Characterization of the major dehydrogenase related to d-lactic acid synthesis in Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293

Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 is a lactic acid bacterium that converts pyruvate mainly to d-(−)-lactic acid by using d-(−)-lactate dehydrogenase (ldhD). The aim of this study was to identify the gene responsible for d-lactic acid formation in this organism and to characterize the enzyme to facilitate the production of optically pure d-lactic acid. A genomic analysis of L. mesenteroides ATCC 8293 revealed that 7 genes encode lactate-related dehydrogenase. According to transcriptomic, proteomic, and phylogenetic analyses, LEUM_1756 was the major gene responsible for the production of d-lactic acid. The LEUM_1756 gene, of 996 bp and encoding 332 amino acids (36.5 kDa), was cloned and overexpressed in Escherichia coli BL21(DE3) Star from an inducible pET-21a(+) vector. The enzyme was purified by Ni-NTA column chromatography and showed a specific activity of 4450 U/mg, significantly higher than those of other previously reported ldhDs. The gel permeation chromatography analysis showed that the purified enzyme exists as tetramers in solution and this was the first report among lactic acid bacteria. The pH and temperature optima were pH 8.0 and 30 °C, respectively, for the pyruvate reduction reaction, and pH 11.0 and 20 °C, respectively, for the lactate oxidation reaction. The Km kinetic parameters for pyruvate and lactate were 0.58 mM and 260 mM, respectively. In addition, the kcat values for pyruvate and lactate were 2900 s−1 and 2280 s−1, respectively. The enzyme was not inhibited by Ca2+, Co2+, Cu2+, Mg2+, Mn2+, Na+, or urea, but was inhibited by 1 mM Zn2+ and 1 mM SDS.