Enzymatic synthesis of glutathione using yeast cells in two-stage reaction

Abstract

In the present study, permeated yeast cells were used as the catalyst to synthesize glutathione. When waste cells of brewer's yeast were incubated with the three precursor amino acids and glucose for 36 h, 899 mg/L of glutathione were produced. To release the feedback inhibition of gamma-glutamylcysteine synthetase caused by glutathione, two-stage reaction was adopted. In the first stage, glycine was omitted from the reaction mixture and only gamma-glutamylcysteine was formed. Glycine was then added in the second stage, and 1,569 mg/L of glutathione were produced. The conditions of the two-stage reaction were optimized using Plackett-Burman design and response surface methodology. Under the optimized condition, commercially available baker's yeast produced 3,440 mg/L of glutathione in 30 h, and most of the produced glutathione was in the medium. The two-stage reaction could effectively reduce the feedback inhibition caused by glutathione, but degradation of glutathione was significant.

Systematic manipulation of glutathione metabolism in Escherichia coli for improved glutathione production

“…Additionally, the intracellular GSH concentration of the strain carrying the mutant GSH I was 1.3-fold higher than that of the control [ 8 ]. The method of two-stage reaction was also adopted to release the feedback inhibition of GSH I caused by glutathione, and under the optimized condition, commercially available baker’s yeast produced 3.44 g/L of glutathione in 30 h [ 9 ]. A novel enzyme (the bifunctional glutathione synthetase encoded by gshF ) that possessed both γ-GCS and GS activities and was non-sensitive to GSH was discovered in several microorganisms, including Streptococcus agalactiae [ 10 ], Enterococcus faecalis [ 10 ], Listeria monocytogenes [ 11 ], and Streptococcus thermophiles [ 12 ].…”

One-pot synthesis of glutathione by a two-enzyme cascade using a thermophilic ATP regeneration system

“…When GSH synthesis was performed by using whole-cell catalysis in S. cerevisiae, intracellular ATP generation system was generally used. Due to the low efficiency of ATP generation and enzymatic activity of GSH synthetase, the titer and productivity of GSH were not satisfactory (Li et al, 2010). In the present in vitro two-enzyme system, the overall GSH production was improved under the optimal condition.…”

Production of glutathione using a bifunctional enzyme encoded by gshF from Streptococcus thermophilus expressed in Escherichia coli

“…4B and C. The glycolytic pathway in S. cerevisiae is widely used for ATP generation to be coupled with reactions requiring ATP. However, only a small part of generated ATP was used for the ATP-requiring GSH synthesis reaction (Murata et al, 1981); most seemed to be hydrolyzed to generate a great amount of heat (Li et al, 2010). In order to establish an effective ATP generation system, we examined a series of reaction systems by coupling the reaction using induced JM109 (pTrc99A-gshF) with 19 mutant strains of S. cerevisiae BY4742, in each of which a single gene related with ATP hydrolysis had been deleted (Table 1).…”

“…We previously used a two-stage reaction mode in which glycine was not added in the first stage of reaction to allow the formation of more ␥-GC, and then glycine was added in the second stage to form GSH. In such a way, 3.44 g l −1 were produced in 30 h and most of the GSH was in the medium (Li et al, 2010).…”