Gamma-glutamylcysteine synthesis in the yeast Yarrowia lipolytica and its application as an antioxidant in food materials

In this thesis, we improve γGC production in the yeast Y. lipolytica primarily by deletion of the biosynthetic GSH2, which encodes the glutathione synthetase (GSS) enzyme, and by overexpression of the GSH1 gene, together with MET4 which are thought to have an effect on cysteine anabolism. DG2-based strains (DG2G1, DG2G1M4, DG2-2G1, DG2-2G1M4) were particularly effective at boosting thiol production compared to the WT strain, with the M4 modification enhanced this effect. Antioxidant and antimicrobial properties of γGC and GSH were tested. According to DPPH test, IC50 values were 0.29 mM and 0.36 mM for γGC and 0.19 mM and 0.22 mM for ABTS, respectively. According to agar diffusion test, both γGC and GSH were ineffective against Escherichia coli growth, while the zone of inhibition for Listeria monoycytogenes, Staphylococcus aureus and Bacillus cereus was 15.20, 15.11, 12.95, 12.31 and 11.67, and 9.2 mm for γGC and GSH, respectively. The minimum inhibition concentration was determined as 10 mM, 5 mM, 10 mM, 5 mM, 5 mM for GC and 5 mM, 5 mM, 10 mM, 10 mM, 10 mM for GSH in Escherichia coli, Listeria monoctogenes, Staphylococus aureus and Bacillus cereus, respectively.In the last part of the study, we chose two food products, sunflower oil and wine, which are prone to oxidative degradation, to see the antioxidant effect of GSH and γGC, in foods. The samples resistance to the generation of primary and secondary oxidation products was T>GCT>γGC>GSHT>GSH>C for up to 15 days under storage conditions. The antioxidant effect of two thiols, γ-glutamylcysteine and glutathione, in sunflower oil under accelerated storage was studied. Oil samples were stored at 50°C for a period of 15 days. The samples resistance to the generation of primary and secondary oxidation products was T>GCT>γGC>GSHT>GSH>C for up to 15 days under storage conditions. White wine were maturated for two months following the separate addition of SO2, γGC, and GSH at the concentration of 30 mg/L. As a result, the protective properties of γGC and GSH additives on phenolic compounds and decelerating the browning degree in white wine were eluciated. At the end of the maturation period, γGC (218,43 GAE/L), GSH (215,22 GAE/L) treatments had the highest amount of phenolic compounds, followed by SO2 (205,57 GAE/L) and control (192,23 GAE/L). Furthermore, γGC (OD420 0.032) added wine displayed almost the same level of browning with the SO2 (OD420 0,031) added wine.