Synthesis Of Glutathione By A Two-enzyme Cascade

Glutathione is a key non-protein thiol compound widely used in the food and pharmaceutical industries.In this study,a two-step reaction for the synthesis of GSH by a GshF enzyme with weak product inhibition was used.The introduction of PPK catalyzes the low-cost polyphosphate and ADP to synthesize ATP,which reduces the production cost.The GshF and PPK were constructed on the bionic carrier,which realized the reuse of the enzyme and prolonged the service life of the enzyme.(1)Constructed from Pasteurella multocida,Streptococcus agalactiae and the enzyme GshF PPK enzymes from Rhodobacter sphaeroides(Rhodobacter sphaeroides),Corynebacterium glutamicum(Corynebacterium glutamicum)of Recombinant expression vector.The correct expression of GshF and PPK enzymes was achieved,and the expression levels were all 4-5 mg/mL.(2)The reaction conditions for the production of GSH system by double enzymatic method were optimized,and a series of optimal reaction conditions were obtained:GshF(Sa),PPK(Rs),T(37?),pH(9.0),ATP.Initial addition amount(5%),PolyP concentration(P6:Cys=1.5:1),GshF and PPK enzyme ratio(1:1),and amino acid ratio(3:1:3).And the substrate concentration of the GSH production system was expanded from 30 mM to 50 mM.Under the optimal conditions,the production of GSH increased from 8.76g/L to 13g/L,and the corresponding conversion rate was 86.36%.The external addition of ATP was only 5%of the theoretical demand of the system,saving 95%.(3)GshF from Streptococcus agalactiae and PPK from Spherical Red Bacteria and polydopamine(PDA)microcapsuLe scaffolds were combined to form a biomimetic fixed double enzyme catalytic system.The effect of the immobilized PPK enzyme was verified,the fixed position of the double enzyme was optimized,and the system showed higher enzyme reuse.37?,350 rpm under nitrogen gas protection conditions,the bionic immobilized double enzyme can be reused in ten batches and the conversion rate can still reach 75.11%.The system provides a reference scheme for cofactor regeneration,cascade reaction(cycle reaction),and protection of multi-subunit enzyme activity.