title: Rationally re-designed mutation of NAD-independent L-lactate dehydrogenase: high optical resolution of racemic mandelic acid by the engineered Escherichia coli.
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.
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China.
authors.forename: Tianyi
authors.initials: T
authors.lastname: Jiang
authors.forename: Chao
authors.initials: C
authors.lastname: Gao
authors.forename: Peipei
authors.initials: P
authors.lastname: Dou
authors.forename: Cuiqing
authors.initials: C
authors.lastname: Ma
authors.forename: Jian
authors.initials: J
authors.lastname: Kong
authors.forename: Ping
authors.initials: P
authors.lastname: Xu
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D004926:Escherichia coli / Q000378:metabolism*; D005818:Genetic Engineering; D006038:Glyoxylates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D008333:Mandelic Acids / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009904:Optical Rotation; D044223:Pseudomonas stutzeri / Q000201:enzymology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D016415:Sequence Alignment; D013237:Stereoisomerism; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D006038:Glyoxylates; D008333:Mandelic Acids; D011994:Recombinant Proteins; C012482:phenylglyoxylic acid; D007770:L-Lactate Dehydrogenase; C037938:mandelic acid