Study On The Glutathione Synthesis And Energy Strategy In Enzymatic Process

The precursor compounds of reduced glutathione(glutathione,GSH)are cysteine,glycine and glutamic acid,and its molecular structure has two kinds of active groups,gamma glutamyl and sulfhydryl groups.Glutathione is more and more widely used in the healthcare industry,food and daily chemical industry,besides it is expensive.Enzymatic synthesis of GSH provide a new way for the industrial production of GSH.At present,there are few reports on enzymatic synthesis of GSH,and the effective supply of energy in the process of reaction is an important problem to be solved by enzymatic synthesis of GSH.The main research contents and results are as follows:(1)Optimization of enzymatic synthesis of GSH.The GSH conversion rate was used as response value,response surface methodology was used to investigate four important factors affecting the enzymatic synthesis of GSH(glucose content,amount of bacteria,enzyme catalyzed time and concentration of magnesium chloride),and to determine the optimal conditions for enzymatic synthesis of GSH by RSM method:glucose concentration was 105 mmol/L,the biomass was 2.12 g,the reaction time is5.9 h,concentration of magnesium chloride 10 mmol/L.Under this condition,the conversion rate of GSH reached 27.35%,and compared with the response surface prediction results is 27.41%,the relative error was 0.21%.The result of response surface experiment is reliable.(2)Through the comparison of the types of sugar used and the regulation of energy strategy added in the reaction process for cell enzyme catalyzed synthesis of glutathione(GSH)from the precursor amino acids,the effects of energy types and added modes on the conversion efficiency of enzymatic synthesis of GSH have been investigated.The results showed that,the glucose as an energy carbon source had obvious effect on the enzyme catalyzed synthesis of GSH by yeast cells,adding 100mmol/L glucose in the initial solution,the concentration of GSH synthetised in enzymatic reaction solution was 5.58 mmol/L,the conversion rate of enzyme catalyzed synthesis of GSH was 27.88%,and using batch addition glucose operation(adding 27.78 mmol/L glucose at 2,3,4 hours,respectively)in the initial reaction solution,the conversion rate of cell enzyme catalyzed synthesis of GSH could reach35.81%.In the energy supply strategy of enzyme reaction process,the conversion rate of enzyme catalyzed synthesis of GSH increased to 36.37% when adding 0.5 g/L adenosine in the initial reaction solution.When the energy strategy adopted adding0.5 g/L adenosine combined with glucose batch feeding(adding 27.78 mmol/L glucose at 2,3,4 hours,respectively)in the initial reaction solution,the high spot mass concentration of GSH synthetised in enzymatic reaction solution was 8.31 mmol/L,the conversion rate of GSH reached to 41.57%,which was higher than that of control group by 49.10%.(3)Optimization of precursor amino acid adding mode of experiment,using three kinds of precursor amino acids of synthetic GSH and the energy substance glucose in the reaction fluid as the research object,the amino acid concentration of the three precursors was investigated and the effect of adding different concentrations of amino acids at different times on the enzymatic synthesis of GSH.The experimental results show that the initial precursor amino acid content is 15 mmol/L,the synthesis of GSH was relatively high,at second hours,6 mmol/L of glutamic acid was added,and 6 mmol/L of cysteine was added at third hours,and 8 mmol/L glycine was added at fourth hours,which can effectively increase the content of GSH.Then the design of single point,two,three amino acid added,the results show that the three points with GSH were significantly higher than that of single point and two points,namely,At second hours,6 mmol/L of glutamic acid was added,6 mmol/L cysteine and 83.33 mmol/L glucose was added at third hours,and 8 mmol/L glycine was added at fourth hours,the result is the best.GSH content can reach 8.13 mmol/L,the concentration of GSH than the control group,the initial reaction solution(5.58mmol/L)increased by 51.43%.(4)Experimental research on GSH two phase synthesis.The experimental study on reaction process of delayed addition of glycine,in the first,the reaction system synthesis of gamma glutamyl cysteine,glycine and then add to the reaction liquid,in order to reduce the GSH of gamma glutamylcysteine synthetase(GSH 1)inhibition,improve the conversion rate of GSH.The effect of GSH concentration on GSH I activity had investigated,found that when the GSH concentration reached 5.54mmol/L,which will have a strong inhibitory effect on GSH synthesis enzyme.While precursor amino acid concentration was 10 mmol/L,the conversion rate of GSH reached the highest,and the precursor amino acid concentration was 20 mmol/L,the synthesis mass of GSH was the highest,reached 5.58 mmol/L;The effect of delayed addition of glycine on the conversion of GSH was investigated.It was found that the conversion rate of GSH could be obviously increased by adding glycine at third hours.The precursor amino acids and glucose were put into the reaction liquid in twostages(first stage: Glu 20 mmol/L,Cys 20 mmol/L,glucose 50 mmol/L;the second stage: Gly 20 mmol/L,50 mmol/L glucose)on the synthesis of GSH,the final GSH concentration reached 6.83 mmol/L,the conversion rate of GSH was 34.17%,and compared with the control group(27.88%),it was increased by 22.56%.