title: Recombinant expression, purification and characterisation of the native glutamate racemase from Lactobacillus plantarum NC8.
abstract: 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 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.
authors.affiliation: University of Hohenheim, Institute of Food Science and Biotechnology, Department of Biotechnology, Germany.
authors.forename: Nico
authors.initials: N
authors.lastname: Böhmer
authors.forename: Andreas
authors.initials: A
authors.lastname: Dautel
authors.forename: Thomas
authors.initials: T
authors.lastname: Eisele
authors.forename: Lutz
authors.initials: L
authors.lastname: Fischer
mesh_terms: D000591:Amino Acid Isomerases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D019149:Bioreactors; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D048191:Lactiplantibacillus plantarum / Q000201:enzymology*; D008970:Molecular Weight; D013696:Temperature
chemical_list: D018698:Glutamic Acid; D000591:Amino Acid Isomerases; C062292:glutamate racemase