Enzymological characterization of a novel d-lactate dehydrogenase from Lactobacillus rossiae and its application in d-phenyllactic acid synthesis.

A novel lactate dehydrogenase gene, named lrldh, was cloned from Lactobacillus rossiae and heterologously expressed in Escherichia coli. The lactate dehydrogenase LrLDH is NADH-dependent with a molecular weight of approximately 39 kDa. It is active at 40 °C and pH 6.5 and stable in a neutral to alkaline environment below 35 °C. The kinetic constants, including maximal reaction rate (V_max), apparent Michaelis–Menten constant (K_m), turnover number (K_cat) and catalytic efficiency (K_cat/K_m) for phenylpyruvic acid were 1.95 U mg⁻¹, 2.83 mM, 12.29 s⁻¹, and 4.34 mM⁻¹ s⁻¹, respectively. Using whole cells of recombinant E. coli/pET28a-lrldh, without coexpression of a cofactor regeneration system, 20.5 g l⁻¹d-phenyllactic acid with ee above 99% was produced from phenylpyruvic acid in a fed-batch biotransformation process, with a productivity of 49.2 g l⁻¹ d⁻¹. Moreover, LrLDH has broad substrate specificity to a range of ketones, keto acids and ketonic esters. Taken together, LrLDH is a promising biocatalyst for the efficient synthesis of d-phenyllactic acid and other fine chemicals. [ABSTRACT FROM AUTHOR]