Cloning, Expression, Characteristics And Application Of Bifunctional Glutathione Synthetase

As the most abundant non-protein thiol compound in organisms, glutathione is a tripeptide formed from glutamate, cysteine and glycine, and has important biological activity. Therefore it has been widely used in medicine, food and cosmetic. The current production of glutathione in industry is fermentation of Saccharomyces cerevisiae. In this work, a bifunctional glutathione synthtase (GshF) from Listeria monocytogenes was cloned and expressed, the characterization of GshF was studied.Meanwhile, the polyphosphate kinase (PPK) from E. coli K-12 was cloned and expressed to enzymatic synthesis of ATP, then the PPk was used as an ATP regenerator to produce glutathione.The gene coding GshF in Listeria monocytogenes was cloned and linked with vector pET28a(+), resulting in the construction of pET28a(+)-gshF. By adopting E.coli BL21(DE3) to express the exogenous protein GshF, the effects of different expression conditions were investigated to improve the expression level. The highest activity of GshF(2318 U/L) in flasks was achieved when 0.2 mM IPTG was added into the cell culture(in the stage of mid-log growth phase) and induced at 28 ℃ for 6 hours.Due to the His-tag of GshF expressed in E.coli BL21(DE3)/pET28a(+)-gshF, Ni-NTA affinity chromatography was used to purify GshF, the specific activity of purified GshF was 14.68 U/mg which was 15.1 times of that in crude cell lysate, with a good recovery rate of 76%. The characterization of GshF was also studied, the optical catalytic pH of purified GshF was 8.5, and the optimum temperature was 37℃, the optimum concertation of ATP and Mg2+ were 10 mM and 30 mM. Excess ATP would inhibit the GshF. At last, the production of glutathione enzymatic synthesized with purified GshF in those optimum conditions was 2.58 g/L.The fed-batch fermentation of recombinant E.coli BL21(DE3)/pET28a(+)-gshF was carried out with glycerol as the only carbon source, finally, the OD600 reached 86 with 5.6g/L of GshF.The gene codding polyphosphate kinase (PPK) in E.coli K-12 was cloned and expressed in E.coli BL21(DE3). The specific activity of purified PPK after Ni-NTA affinity chromatography was 2.6 U/mg with the recovery rate of 41%. PPK was used to convert ADP to ATP in the presence of PolyP, the rate of ATP/ADP was 1/0.77 when the reaction reached its equilibrium. At last, the purified PPK was used as an ATP regenerator to produce glutathione coupled with GshF, after 3 hours,1.38 g/L glutathione was achieved in the presence of 5 mM ADP and 5 mM Polyp.