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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

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...

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Detalles Bibliográficos
Autores principales: Jiang, Tianyi, Gao, Chao, Dou, Peipei, Ma, Cuiqing, Kong, Jian, Xu, Ping
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3526519/
https://www.ncbi.nlm.nih.gov/pubmed/23176608
http://dx.doi.org/10.1186/1475-2859-11-151
Descripción
Sumario: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.