Directed Evolution Of Glutathione Bifunctional Synthase And Its Application In L-Glutathione Synthesis

L-Glutathione(GSH)is a small peptide with many important physiological functions and has an increasing market potential.Although the production of glutathione has been achived with industrial yeasts,there are still some problems,such as strong inhibition caused by intracellular product concentration,and high cost of product purification.The purpose of this project is to develop a cheap and efficient process for in vitro enzymatic synthesis of GSH.Firstly,the glutathione bifunctional synthetase gene(gshFSA)in Streptococcus agalactiae was cloned and transformation to E.coli,then the enzymatic properties were examined extensively with the purified recombint enzyme.With a high-throughput screening platform based on arsenic ion pressure on cell growth,the enzyme activity of the GshFSA was improved by directed evolution and screening.On the other side,recombinant Acetate Kinase(ACK)was produced and used to construct an ATP regeneration system,which was employed to construct dual-enzyme(GshFSA-ACK)coupled system for GSH biosynthesis.The glutathione bifunctional synthetase gene(gshFSA)was amplified by PCR from S.agalactiae,and the recombinant expression vector pET28a-gshFSA was constructed and transformed into E.coli BL21(DE3)to obtain the recombinant cells.The expression conditions were optimized and summarized in the following:induction in the middle period of logarithmic growth(3.5 h,OD600 ? 1.5),0.2 mM IPTG,cell collection after 8 h induction at 25 ?.Under the above conditions the enzyme activity was 293 U/L.With Ni-NTA affinity chromatography,GshFSA can be effectively obtained with the high recovery rate(70.8%)and specific enzyme activity(1.734 U/mg),which was 13.6 times that of the crude enzyme.The conditions for the catalytic reaction were further optimized,and the optimal conditions(37?,pH 8.0)were determined.A gshA-deficient strain(E.coli W3110(?gshA))was constructed by using Red homologous recombination,and a high-throughput screening platform based on arsenic ion pressure was established to screen the strain with high GshFSA bioactivity.The mutation gene library was constructed using error-prone PCR,and five positive mutants(M1-M5)were screened out with siginificant increase in specific enzyme activity and Km(Glu).The specific enzyme activity of mutant M5 was increased by 117.9%to 3.77 U/mg,and Km(Glu)was reduced to about 1/3 of the wild type.Further studies showed that the optimum temperature and pH of the mutant M5 were almost the same as before,and still insensitive to the feedback inhibition of GSH.However,the kcat/Km of the mutant M5 increased from 2.41 s-1mM-1 to 8.59s-1mM-1.The recombinant ACK was produced by cloning the acetate kinase gene from E.coli K-12 and the expression plasmid pET28a-ack was constructed.After induced expresssin and Ni-NTA affinity purification,the recombinant ACK was obtained with high specific enzymatic activity(114.5 U/mg),which is 14.08 times that of the crude enzyme in the broth.This ACK can be used to catalyze ADP to regenerate ATP with the conversion efficiency of ADP(94.6%).This ACK-mediated ATP regeneration system was coupled in a GshFSA-catalyzed synthesis of GSH reaction system.In a 1 mL above reaction system,GSH concentration reached 3.09 g/L after 60 min,and the number of ATP regenerative cycles was approximately 3.02.Finally this two-enzyme system was scaled up to 1 L reaction system,GSH concentration was improve up to 12.64 g/L(41.13 mM)after 3 h,and the productivity of GSH was 4.21 g/(L·h).In the meanwhile,the conversion rate of cysteine to GSH reached 51.4%(mol/mol),and the number of ATP regenerative cycles was improved to 7.25.