High production of glutathione by in vitro enzymatic cascade after
The cell free system has been paid more attention due to its potential
of facilitating more eﬃcient catalysis of multistep reactions. In this
study, an efficient enzymatic cascade of 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
163.3-fold, and its activity raised 18 %. 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, 99.9 mM GSH and 7.6 mM oxidized
glutathione (GSSG) were produced within 2 h. The molar yield was 95.9
mol/mol based on the amino acid added, while the productivities of GSH
achieved 49.95 mM/h, which was the highest yield and productivity ever
reported about GSH synthesis.