Abstract:
Glutathione (GSH), one of the thiol short peptide in organisms, is widely used in medicine, food additive, health care and cosmetic industries. In this study, a two-enzyme cascade consisting of bifunctional glutathione synthetase (GshF) and polyphosphate kinase (PPK) was constructed for ATP-regenerating synthesis of GSH. As the catalytic substrate of PPK, polyphosphate (polyP) was used to provide energy in the reaction. The efficient soluble expressions of GshF and PPK were achieved when induced at a low temperature of 18 °C. After purification, the two enzymes were coupled together to synthesize GSH in one pot. It was found that high concentration of MgCl
2 inhibited the production of GSH due to the chelation with polyP. The optimal concentration ratio of polyP to MgCl
2 was 30∶45. At 0.5 mmol/L ADP, the reaction achieved the highest efficiency for GSH formation. The optimal temperature for the two-enzyme reaction was 45 °C, achieving a high yield of 78% based on added
L-cysteine. The optimal temperature for GSH synthesis was relatively high, because the activities of GshF and PPK could increase with temperature. The enzyme ratio in the system was further investigated. The production of GSH was improved at higher concentration of PPK, indicating that the ATP-regenerating reaction was a limit step in the reaction. At the optimal ratio of 4 g/L PPK and 1 g/L GshF, the titer of GSH reached (58 ± 3.3) mmol/L with a productivity of (19.3 ± 1.1) mmol/L/h at 3 h, which achieved the reported highest production of GSH. Using the low-cost substrate of polyP to supply energy, the efficient cascade reaction provided a potential application for large-scale enzymatic production of GSH.