High production of glutathione by in vitro enzymatic cascade after thermostability enhancement

Abstract

The cell free system has been paid more attention due to its potential of facilitating efficient catalysis of multistep reactions. In this study, an efficient enzymatic cascade of glutathione (GSH) production was developed through the evolution of bifunctional glutathione synthetase (GshF), coupled with polyphosphate kinase (PPK). First, the stability and activity of GshF were enhanced by loop interchange and site‐directed mutagenesis. As a result, the GshF half‐value period increased 163.32‐fold, and its activity raised 18.16%. PPK from Jhaorihella thermophile (PPKJT) was characterized and used to regenerate ATP in the GSH synthesis, with hexametaphosphate (PolyP (6)) as the phosphate donor. After the process optimization, 99.92 mM GSH and 7.61 mM oxidized glutathione (GSSG) were produced within 2 hr. The molar yield was 0.96 mol/mol based on the amino acid added, while the productivities of GSH achieved 49.95 mM/hr, which were the highest yield and productivity ever reported about GSH synthesis.

Programming Integrative Multienzyme Systems and Ionic Strength For Recyclable Synthesis of Glutathione

“…The plasmid pET28a-M3 was constructed previously. 19 The sequence of SpyCatcher/ SpyTag with the flexible linker (GGGGS) 2 was synthesized by Sangon (Shanghai, China). The plasmids pET28a-SpyCatcher-(GGGGS) 2 / pET28a-(GGGGS) 2 -SpyTag with the flexible linker (GGGGS) 2 were amplified with a primer pair of C-F C-R and T-F T-R. GshF was amplified with a primer pair of GshF-F and GshF-R, while PPK was amplified with a primer pair of PPK-F and PPK-R. PCR products were recovered and then cloned into pET28a by homologous recombination for pET28a-SpyCM3 (pET28a-SpyCatcher-(GGGGS) 2 -GshFM3) and pET28a-JTSpyT (pET28a-PPKJT-(GGGGS) 2 -SpyTag) construction.…”

“…Enzymatic Activity and Thermostability Assay. The enzymatic activities of M3 and SpyCM3 were assayed according to the method of Cui et al 19 One unit of enzyme activity was defined as the amount of enzyme that produced 1 μmol GSH per minute.…”

“…16−18 Enzymatic production of glutathione (GSH) has the advantages of higher yield, which has been previously reported in amounts up to 99.9 mM by coupling of GshF and PPK. 19 But the reusability of the MESs is important in industrial-scale enzymatic production of GSH.…”

“…Glutathione, which is synthesized from l -glutamate, l -cysteine, and glycine, is a short peptide. It has already attracted wide attention from food and beverage industries that used it as an aroma stabilizer, flavor enhancer, antioxidant, and a baking agent. − Enzymatic production of glutathione (GSH) has the advantages of higher yield, which has been previously reported in amounts up to 99.9 mM by coupling of GshF and PPK . But the reusability of the MESs is important in industrial-scale enzymatic production of GSH.…”

“…As a source of energy, the cofactor adenosine triphosphate (ATP) is also required for GSH biosynthesis. Recently, the family-2 polyphosphate kinase (PPK2) has already attracted much interest for ATP regeneration, and the substrate polyphosphate (PolyP) shows the advantages of low cost and high stability. , In our previous study, the thermostability and activity of a bifunctional glutathione synthetase (GshF) mutant (M3), which was generated by DNA-shuffling and site-directed mutation, were improved at 45 °C. Thus, the mutant M3 was a better choice for GSH synthesis.…”