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

Tianyi Jiang1, Chao Gao1, Peipei Dou12, Cuiqing Ma1*, Jian Kong1 and Ping Xu12*

Author Affiliations

1 State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100, China

2 State Key Laboratory of Microbial Metabolism and Schoolof Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China

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Microbial Cell Factories 2012, 11:151  doi:10.1186/1475-2859-11-151

Published: 23 November 2012



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.


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.


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.

NAD-independent L-lactate dehydrogenase; Site-directed mutagenesis; Optical resolution; D-mandelic acid