468100 Biochemical Characterization of the N-Acetylglutamate Kinase (ArgB) from Lactobacillus Plantarum
Probiotics, specifically Lactobacillus, offer the opportunity to deliver arginine-producing bacteria to mammalian gastrointestinal tracts. Arginine biosynthesis in prokaryotes is encoded by the arg operon and involves eight enzymatic steps starting at glutamate. The second step is catalyzed by a N-acetylglutamtate kinase, encoded by the argB gene, that phosphorylates an acetylated glutamate molecule. Based on literature, arginine exhibits feedback inhibition toward the ArgB enzyme, which is one of the key methods in how the cell regulates the production of arginine in many organisms. In order to increase the production of arginine, this feedback inhibition must be fully understood and characterized.
The argB gene was isolated from Lactobacillus plantarum, cloned into the pET32a(+) plasmid with a C-terminal His6 tag, and transformed into E. coli BL21(DE3) for expression. The resultant recombinant proteins were recovered from cell lysates by affinity chromatography using a Ni-NTA column and subjected to full biochemical characterization. In future work, results will be used to relieve arginine feedback inhibition of ArgB and enable the construction of arginine-producing L. plantarum.