Study On The Enzymatic Properties And Application Of ?-glutamylcysteine Synthase

?-glutamyl-cysteine is a dipeptide with both?-glutamyl and active hydroxyl groups,which has important physiological functions such as anti-oxidation,detoxification,maintaining the balance of intracellular sulfhydryl groups,transmitting quotation marks and protecting cells.Therefore,?-glutamyl-cysteine has a wide range of application prospects in the fields of food,health care products,cosmetics and medicine.As a bacterial enzyme,?-glutamyl-cysteine synthetase plays an irreplaceable role in the synthesis of?-glutamyl-cysteine and glutathione.In this paper,?-glutamyl-cysteine synthetase was induced and expressed from the genetically engineered strain p ET-28a-Gsh1 derived from E.coli BL21(DE3).Using sodium glutamate and cysteine as substrates,?-glutamyl-cysteine was synthesized by enzymatic method.Main research content and results:1.Optimizing the induced expression and purification of the recombinant genetically engineered strain p ET-28a-Gsh1 derived from E.coli BL21(DE3)and SDS-PAGE analysis.The results showed that at 26?,220 r/min,10 g/L Tianda No.1 inducer that was made by our laboratory was used to induce the recombinant genetically engineered strain p ET-28a-Gsh1 derived from E.coli BL21(DE3)for 12 h.The final purified?-glutamyl-cysteine synthase was as high as 8.5 mg/L,which was about 1.3 times of IPTG inducer.2.The enzymatic properties of?-glutamyl-cysteine synthase were analyzed.The results showed that the optimal catalytic temperature of?-glutamyl-cysteine synthase was40?;the optimal was p H=7.5;the optimal surfactant was 0.09%SDS;the optimal metal ion was 12 mmol/L Fe²⁺+19 mmol/L Na⁺;Fe²⁺can not only relieve the inhibition of glutathione on the activity of?-glutamyl-cysteine synthase,but also increase the activity of the enzyme by 12%.3.The synthesized product was preliminarily separated and purified,the synthesized?-glutamyl-cysteine was analyzed qualitatively and quantitatively by HPLC.The results showed that under the conditions of optimal enzymatic properties and ATP energy supply,the yield of?-glutamyl-cysteine was 33.5 g/L and substrate conversion rate reaches 68.3%.4.Established enzyme method combined with yeast coupling system to regenerate ATP and the reaction system was optimized.The results showed that the optimal ratio of yeast to cetrimonium bromide was 15:1 and the optimal time for cetrimonium bromide to permeate yeast was 4 h;the optimal ratio of raw material glutamic acid to cysteine in the reaction system was 4:5;the optimal concentration of?-glutamyl-cysteine synthase crude enzyme solution was 70 mg/L;the optimal phosphate concentration was 40 mmol/L;the optimal carbon source was glucose and the optimal initial glucose concentration was 220mmol/L,55 mmol/L glucose was added for 5 hours.The final yield of?-glutamyl-cysteine was 12.16 g/L and substrate conversion rate is about 40%.