Abstract :
[en] Gamma-glutamyl cysteine (γGC) is the precursor of glutathione (GSH), the most abundant thiol compound in the cell, which plays an important role in many cellular mechanisms. Applications of GSH in medicine, food, cosmetics and biotechnology have long been known. Recent studies have also shown therapeutic potential of γGC. Y. lipolytica is an unconventional yeast and is of interest as a cell factory for pharmaceutical, food and feed applications.
In this thesis, we improve γGC production in Y. lipolytica primarily by deletion of the biosynthetic GSH2, which encodes the glutathione synthetase (GSS) enzyme, and by overexpression of the GSH1 gene, which encodes the γ-glutamylcysteine ligase (GCL) enzyme, together with MET4, GDH, CYSE and CYSF genes, which are thought to have an effect on the glutamine and cysteine anabolism. Within 24 hours of cell culture, a γGC titre of 464 nmol mg-1 protein (93 mg gDCW-1) was attained using this approach.
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 E. 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 γGC and 5 mM, 5 mM, 10 mM, 10 mM, 10 mM for GSH in E. 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-glutamyl cysteine 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.
