A novel D-mandelate dehydrogenase used in three-enzyme cascade reaction for highly efficient synthesis of non-natural chiral amino acids

J Biotechnol. 2015 Feb 10;195:67-71. doi: 10.1016/j.jbiotec.2014.10.026. Epub 2014 Oct 27.

Abstract

A novel NAD(+)-dependent D-mandelate dehydrogenase was identified from Lactobacillus brevis (LbDMDH). After purified to homogeneity, the optimum pH and temperature for oxidation of D-mandelate were pH 10.0 and 40 °C, and the Km and kcat were 1.1 mM and 355 s(-1) respectively. Employing the LbDMDH together with a mandelate racemase from Pseudomonas putida and a leucine dehydrogenase (EsLeuDH) from Exiguobacterium sibiricum, we established a three-step one-pot domino reaction system for preparing chiral L-phenylglycine from racemic mandelic acid with internal cofactor recycling. Under the optimum conditions, 30.4 g rac-mandelic acid (0.2 M) at 1L scale had been converted into chiral L-phenylglycine, with 96.4% conversion, 86.5% isolation yield, >99% eep and 50.4 gL(-1)d(-1) space-time yield.

Keywords: Asymmetric synthesis; Cascade reaction; D-Mandelate dehydrogenase; L-Phenylglycine; Process optimization.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alcohol Oxidoreductases / chemistry
  • Alcohol Oxidoreductases / isolation & purification
  • Alcohol Oxidoreductases / metabolism*
  • Bioreactors
  • Fungal Proteins / chemistry
  • Fungal Proteins / isolation & purification
  • Fungal Proteins / metabolism*
  • Glycine / analogs & derivatives*
  • Glycine / chemistry
  • Glycine / metabolism
  • Lactobacillus / enzymology
  • Mandelic Acids / metabolism
  • Pseudomonas putida / enzymology
  • Racemases and Epimerases / chemistry
  • Racemases and Epimerases / isolation & purification
  • Racemases and Epimerases / metabolism*
  • Stereoisomerism

Substances

  • Fungal Proteins
  • Mandelic Acids
  • 2-phenylglycine
  • Alcohol Oxidoreductases
  • D-mandelate dehydrogenase
  • Racemases and Epimerases
  • mandelate racemase
  • mandelic acid
  • Glycine