Purification of extra cellular poly-γ-glutamic acid as an antibacterial agent using anion exchange chromatography

BLAST analysis of the 16S rRNA gene sequence for the newly isolated bacterium, revealed significant identity (99.5%) with Bacillus sonorensis [Ijadi Bajestani, M., et al., International Journal of Biological Macromolecules, 2017. 96: p. 100-110]. According to the literature review for closely related species of Bacillus sonorensis, the production of poly-γ-glutamic acid (γ-PGA) as an extra cellular biopolymer was investigated for the isolated bacteria which is deposited in IBRC (Iranian Biological Resource Center) as Bacillus sp. Strain M2 (IBRC-M11173). To determine if γ-PGA production by Bacillus sp. Strain M2 is glutamate dependent, it was grown on PGA medium, consisted of sodium glutamate. The results proved that γ-PGA production is highly dependent on glutamate component. In the following, the bioproduct has undergone different purification processes mainly consisting of dialysis, deproteinization and anion exchange chromatography. Based on the high-performance liquid chromatography (HPLC) results for ion chromatography effluents, 59% of the initial PGA in main solution was eluted via NaCl elution. Gel permeation chromatography (GPC) characterization analysis was accomplished to determine the polydispersity and γ-PGA molecular weight. Two major average molecular weights were distinguished; the heavy weight fraction of 7.7×10⁶g/mol with polydispersity index of 1.73 and the other one with an average molecular weight number of 1.7×10⁴g/mol and polydispersity index of 4.4. The antibacterial activity of the extracellular γ-PGA, as an anionic biopolymer, toward Staphylococcus aureus and E. coli, was assayed using the clinical and laboratory standards institute (CLSI) guidelines. For Staphylococcus aureus the minimum inhibitory concentration (MIC) value was about 34g/L while for E. coli this value reaches 53g/L.