Glutathione and hydrogen peroxide production in Enterococcus italicus: an attractive biotechnological standpoint

Glutathione (GSH, L-γ-glutamyl-L-cysteinylglycine), one of the most abundant non-protein thiol widely distributed in living organisms, including human, yeast, and bacterial cells, plays a significant role in maintaining normal redox environment of cells. For its antioxidant properties, the commercial demand for GSH is expanding in pharmaceutical and food industries (Carmel-Harel and Storz, 2000). Biosynthesis of GSH in cells usually involves two sequential ATP-dependent reactions catalyzed by γ-glutamylcysteine (γ-GC) synthetase (γ-GCS, EC 6.3.2.2) and GSH synthetase (GS, EC 6.3.2.3). In most cells, the two enzymes are encoded by separate genes, gshA and gsh B (gsh1 and gsh 2 in eukaryotes), respectively. The activity of γ-GCS is subjected to feedback inhibition by GSH to avoid over-accumulation of GSH, and it has been shown that formation of γ-GC is the rate-limiting step in the GSH biosynthesis pathway. In contrast to the extensive studies in eukaryotic cells, relatively little is known about glutathione in prokaryotes. A survey of protein database like PFAM and NCBI reveals that genes with high similarity to gshA, but not to gsh B, are found in Gram-positive bacteria, suggesting that these bacteria lack the capability to synthesize glutathione, although able to import GSH from environment. However, evidence exists that some Lactic Acid Bacteria are able to synthesize GSH, in relatively high concentration, as Enterococcus faecalis and Streptococcus thermophilus (Wei et al., 2011). Through the analysis of the recently available genome shotgun sequence of Enterococcus italicus DSM15952T type strain (Accession: PRJNA61487, ID: 61487), we found in this novel enterococcal species isolated from typical Italian cheeses, the presence of the gene encoding for an enzyme possessing both γ-GCS and GS activities. This bifunctional enzyme, termed γ-GCS-GS or GshF, identified in a few of Gram positive bacteria, is reported to remove the feedback inhibition, enhancing GSH biosynthesis (Vergauwen et al., 2006). The aim of present study was to determine the production and accumulation of glutathione in E. italicus, a species that could represent a new safe adjunct culture for the dairy industry (Fortina et al., 2008; Borgo et al., 2009). The study was conducted through different steps: 1) selection of nutrients in the culture medium, 2) response to physiological stress, 3) preparation of cell extracts for protein analysis and GSH detection via enzymatic recycling method and fluorometric HPLC, 4) scale-up in 6 L bioreactor for the enhancement of cell density and intracellular GSH content. It is interesting to note that the intracellular GSH accumulation in E. italicus increased its resistance to hydrogen peroxide, produced by NADH oxidase activity. Genome analysis indicated the presence of a NADH oxidase-H2O2 forming gene (nox1) that can explain the ability of E. italicus to produce high amount of this compound. Hydrogen peroxide is known to have a rapid bacteristatic/bactericidal effect and this characteristic, combined with the production of GSH, makes E. italicus especially attractive for tayloring of cheeses with enhanced flavor and for development of bio-preservation strategies.