Recombinant expression, purification and characterisation of the native glutamate racemase from Lactobacillus plantarum NC8

Protein Expr Purif. 2013 Mar;88(1):54-60. doi: 10.1016/j.pep.2012.11.012. Epub 2012 Dec 8.


Glutamic acid racemases (MurI, E.C. catalyse the racemisation of L- and D-glutamic acid. MurIs are essential enzymes for bacterial cell wall synthesis, which requires d-glutamic acid as an indispensable building block. Therefore these enzymes are suitable targets for antimicrobial drugs as well as for the potential design of auxotrophic selection markers. A high expression system in Escherichia coli BL21 (DE3) was constructed to produce and characterise the biochemical properties of the MurI from Lactobacillus plantarum NC8. In a 4-L-bioreactor cultivation, 3266 nkat(D-Glu)/mg(protein) of specific enzyme activity was produced. The recombinant, tag-free Murl was purified by an innovative affinity chromatography method using L-glutamic acid as the relevant docking group, followed by an anion exchange chromatography step (purification factor 9.2, yield 11%). This two-step purification strategy resulted in a Murl sample with a specific activity of 34,060 nkat(D-Glu)/mg(protein), comprising a single protein band in SDS-PAGE. The purified Murl possessed an assay temperature optimum of 50 °C, but it was not stable at this temperature. The half-lives of the purified Murl were 162 h at 20 °C and only 1.9 h at 40 °C. The Murl activity was maximum between pH 7 and 10, resulting in a maximal half-life of 287 h at pH 7. Only D- and L-glutamic acid were recognised as substrates for the Murl with similar K(cat)/K(M) ratios of 3.6s(-1)/mM for each enantiomer.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Isomerases / chemistry
  • Amino Acid Isomerases / genetics*
  • Amino Acid Isomerases / isolation & purification*
  • Bioreactors
  • Cloning, Molecular
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / genetics
  • Gene Expression
  • Glutamic Acid / chemistry
  • Glutamic Acid / metabolism
  • Kinetics
  • Lactobacillus plantarum / enzymology*
  • Molecular Weight
  • Temperature


  • Glutamic Acid
  • Amino Acid Isomerases
  • glutamate racemase