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

Abstract

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 39kDa. 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, 2.83mM, 12.29s-1, and 4.34mM-1s-1, respectively. Using whole cells of recombinant E. coli/pET28a-lrldh, without coexpression of a cofactor regeneration system, 20.5gl-1 d-phenyllactic acid with ee above 99% was produced from phenylpyruvic acid in a fed-batch biotransformation process, with a productivity of 49.2gl-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.