Enzymatic Synthesis Of Glutathione Coupled With Resin Separation

Glutathione(GSH)is a small molecular three peptide compound composed of glutamic acid,cysteine and glycine.It has two active groups of ?-glutamyl and sulfhydryl groups.Glutathione can maintain normal immune system function,and possesses the functions of anti-oxidation and integrated detoxification.It is widely used to delay body aging,enhance immunity and anti-tumor drugs and foods.However,the glutathione synthetase system is feedback inhibited by glutathione produced.In this paper,glutathione can be separated in time by coupling penetration cells with resin,in order to reduce or relieve the effect of feedback inhibition.The main research contents and experimental results are as follows:(1)Penetration of cells.The Saccharomyces cerevisiae(SP5)were penetrative by physical(freeze-thaw method)and chemical reagent(organic solvent and surfactant)method,and the penetration effects were compared.Single factor experiments on SDS were carried out to investigate the effects of penetration concentration,bacterial liquid ratio,penetration time and penetration temperature on the enzymatic synthesis of GSH from Saccharomyces cerevisiae.Through verification,the optimization process is determined as follows: penetration concentration 2 g/L,bacterial liquid ratio 1: 5,penetration time 30 min,penetration temperature 30?.After penetrative treatment,the total amount of GSH was 415 mg/L and the extracellular yield was 89 mg/L.(2)Two-step enzymatic synthesis of glutathione.This paper examined the effect of optimal cell addition and GSH addition on glutathione synthesis.The paper experiments delayed the production of GSH by retarding the addition of glycine,reduced the feedback inhibition of GSH and increased the yield of GSH.Considering the process and the simple operation,glycine was added at the optimum concentration of 50 mmol/L after the reaction 1 h.Two-step enzymatic synthesis of GSH yielded596 mg/L,an increase of 7.97% compared to the blank control.(3)Optimization of enzymatic synthesis of GSH conditions.In this paper,response surface methodology(RSM)was used to optimize the conditions for the enzymatic synthesis of GSH.RSM was used to determine GSH yield as the response,with glutamic,cysteine,glycine and addition time as the single factor.The optimal conditions for the synthesis of GSH were: glutamic 25 mmol/L,cysteine 15 mmol/L,glycine 50 mmol/L and addition time 1 h.Under this optimal process,the validation test was conducted.The average GSH yield reached 793.33 mg/L,which was close to the regression equation.This model is valid.(4)Enzymatic synthesis of glutathione coupled with resin.In this paper,the adsorption and desorption capacities of three different types of adsorbents,D001 cation exchange resin,D301 anion exchange resin and 001x7 acid exchange resin,resin D301 was finally determined as a preferred coupling adsorbent resin.By single factor and orthogonal experiments investigated the effect of D301 resin on enzymatic synthesis of GSH.The final results showed that add 2 g/10 mL of adsorption resin D301 for 2 h,and add 60% of initial concentration of precursor material after 3 h of reaction,the final yield of GSH reached 1154 mg/L,an increase of 77.5% over the original.Using the two-step method combined with the coupling resin method,a slight increase in yield,the reaction time is prolonged,and the final yield can reach1167 mg/L.