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 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(-1) d-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.