Characterization of Key Enzymes for D-lactic Acid Synthesis in Leuconostoc Citreum KM20

Leuconostoc citreum is a heterofermentative lactic acid bacteria, which converts pyruvate to D-lactic acid using D-lactate dehydrogenase. For analyzing the characteristics of the key enzyme for D-lactic acid synthesis in Leuconostoc citreum KM20, the key gene of D-lactic acid synthesis was identified by genomics and transcriptomic analysis, which was cloned into the vector pET-15b and transformed into Escherichia coli BL21 (DE3). The D-lactate dehydrogenase was purified by Ni-NTA column affinity chromatography and its enzymatic property was studied. The results showed that LCK_00389 gene was the key gene among the seven genes related to lactic acid synthesis. The molecular mass of the enzyme encoded by LCK_00389 was 38.8 kDa; the specific activity was 272.71 U/mg; the optimum pH and temperature for pyruvate reduction were 8.0 and 20°C, those for lactate oxidation were 12.0 and 30°C, respectively; the Km and the kcat value of pyruvate were 0.24 ± 0.01 mmol/L and 165.99 ± 3.65 s−1, those of D-lactic acid were 33.27 ± 2.72 mmol/L and 14.67 ± 0.29 s−1; Ca2+, Cu2+ and Na+ promoted the activity of the enzyme, while Zn2+ and Sodium dodecyl sulfate (SDS) inhibited the activity of the enzyme; in addition to using pyruvate as substrate, it also had had strong catalytic activity for oxaloacetate, phenylpyruvate and 2-ketoglutarate.