Email updates

Keep up to date with the latest news and content from Microbial Cell Factories and BioMed Central.

Open Access Research

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

For all author emails, please log on.

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

Published: 23 November 2012

Abstract

Background

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.

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