Enhancement of the catalytic activity of d-lactate dehydrogenase from Sporolactobacillus laevolacticus by site-directed mutagenesis

Abstract

Sporolactobacillus laevolacticus is a producer of d-lactic acid with high optical purity. The amino acid sequence of d-lactate dehydrogenase (D-LDH) from S. laevolacticus was compared with those of other lactate producers. To enhance the activity of D-LDH from S. laevolacticus, some amino acid residues close to the substrate binding site or the active center were replaced by site-directed mutagenesis. Ala234 of D-LDH from S. laevolacticus was found to be an important amino acid residue that positively affects catalytic activity. Site-saturation mutagenesis of the 234th residue was performed. The mutant D-LDH at the 234th residue from alanine to serine or glycine showed enhanced catalytic activity toward pyruvate. The kinetic analysis revealed that the kcat/Km of D-LDH_A234S and _A234G on pyruvate increased 1.9- and 1.2-fold, respectively. Furthermore, the double mutant D-LDH_T75L/A234S improved kcat/Km by 6.8-fold compared to that of wild-type D-LDH. These results showed the potential of the mutant D-LDH for microbial production of d-lactic acid by heterologous expression.