Assembly Of Multi-enzymes With Protein Scaffolds To Synthesize Glutathione

As Glutathione, which has many physiological functions such as antioxidant, anti-virus, and enhancing the body immunity, is a tripeptide containing a thiol group.it has been widely used in pharmaceutical, cosmetic and food industry. In this study, an artificial celluloses complex was constructed by tethering three enzymes: GshF-Doc1-1、ADK-DocⅡ、PAP-Doc1-3 to a protein scaffold, Which aims at improving the efficiency of synthesis of glutathione.First, the gene coding GshF-Doc1-1, adenylate kinase containing dockerin domain (ADK-DocⅡ) and Polyphosphate AMP Phosphotransferase dockerin domain (PAP-Doc1-3) was cloned and linked with vector pET28a(+), resulting in the construction of pET28a(+)-GshF-Doc1-1, pET28a(+)-PAP-Doc1-3, pET28a(+)-ADK-DocⅡ. Meanwhile, gene Coh1-1-CohⅡ-CBD-Coh1-3 containing three cohein domains of the enzymes was cloned and linked with vector pETDuet1, resulting in the construction of pETDuet-1-Coh1-1-CohⅡ-CBD-Coh1-3.Then,By adopting E.coli BL21(DE3) to express GshF-Doc1-1, the effects of different expression conditions were investigated to improve the expression level. The highest activity of GshF-Doc1-1 (2332 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. Ni-NTA affinity chromatography was used to purify GshF-Doc1-1, the specific activity of purified GshF-Doc1-1 was 13.24 U/mg which was 13.4 times of that in crude cell lysate, with a good recovery rate of 76%. The characterization of GshF-Doc1-11-1 was also studied, the optical catalytic pH of purified GshF-Doc1-1 was 8.5, and the optimum temperature was 37℃, the optimum concertation of ATP and Mg2+ were 10 mM/L and 30 mM/L. Excess ATP would inhibit the GshF-Doc 1-1.The adenylate kinase containing dockerin domain(ADK-DocⅡ) was also expressed in E.coli BL21(DE3). The specific activity of purified ADK-DocⅡ after Ni-NTA affinity chromatography was 2.8 U/mg with the recovery rate of 42.4%. ADK-DocII was used to convert ADP to ATP in the presence of PolyP, the rate of ATP/ADP was 1/1.32 when the reaction reached its equilibrium. The characterization of ADK-DocII was also studied, the optical catalytic pH of purified ADK-DocII was 8.0, and the optimum temperature was 40℃.The Polyphosphate AMP Phosphotransferase containing dockerin domain (PAP-Docl-3) was also expressed in E.coli BL21(DE3). The specific activity of purified PAP-Docl-3 after Ni-NTA affinity chromatography was 2.3 U/mg with the recovery rate of 70.2%. PAP-Docl-3 was used to convert AMP to ADP in the presence of PolyP, the rate of AMP/ADP was 1/1.52 when the reaction reached its equilibrium. The characterization of PAP-Docl-3 was also studied, the optical catalytic pH of purified PAP-Docl-3 was 8.5, and the optimum temperature was 50℃.At last, GshF-Docl-1、ADK-Doc PAP-Docl-3 were tethering to the protein scaffold. The biolayer interferometry sensorgrams verified the formation of the three enzyme complex. Then the complex was used to synthesize glutathione, after 3 hours, 5.6mmol/L glutathione was yielded in the presence of 5 mM ATP and 5 mM Polyp.the yield of glutathione raised 25% than the reaction without protein scaffold.The consumption of 11.2 mmol/L ATP in this process showed that ATP regeneration efficiency was about 2. The optimum temperature of the enzymatic reaction system was 37℃, the optimum pH was 7.5.