Recombinant expression, purification and characterisation of the native glutamate racemase from Lactobacillus plantarum NC8
- Publikations-Art
- Zeitschriftenbeitrag (peer-reviewed)
- Autoren
- Böhmer, N.; Dautel, A.; Eisele, T.; Fischer, L.
- Erscheinungsjahr
- 2013
- Veröffentlicht in
- Protein Expression and Purification
- Verlag
- Elsevier
- Band/Volume
- 88 (1)/
- DOI
- 10.1016/j.pep.2012.11.012
- Seite (von - bis)
- 54 - 60
Glutamic acid racemases (MurI, E.C. 5.1.1.3) 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 nkatd-Glu/mgprotein 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 nkatd-Glu/mgprotein, 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 Kcat/KM ratios of 3.6 s−1/mM for each enantiomer.