Rationally re-designed mutation of NAD-independent l-lactate dehydrogenase: high optical resolution of racemic mandelic acid by the engineered Escherichia coli

NAD-independent l -lactate dehydrogenase ( l -iLDH) from Pseudomonas stutzeri SDM can potentially be used for the kinetic resolution of small aliphatic 2-hydroxycarboxylic acids. However, this enzyme showed rather low activity towards aromatic 2-hydroxycarboxylic acids. Results Val-108 of l -iLDH was changed to Ala by rationally site-directed mutagenesis. The l -iLDH mutant exhibited much higher activity than wide-type l -iLDH towards l -mandelate, an aromatic 2-hydroxycarboxylic acid. Using the engineered Escherichia coli expressing the mutant l -iLDH as a biocatalyst, 40 g·L -1 of dl -mandelic acid was converted to 20.1 g·L -1 of d -mandelic acid (enantiomeric purity higher than 99.5%) and 19.3 g·L -1 of benzoylformic acid. Conclusions A new biocatalyst with high catalytic efficiency toward an unnatural substrate was constructed by rationally re-design mutagenesis. Two building block intermediates (optically pure d -mandelic acid and benzoylformic acid) were efficiently produced by the one-pot biotransformation system.