Enzymatic Synthesis And Determination O F Glutathione

In this paper, the enzymatic synthesis and determination of glutathione(GSH) has been investigated, its research contents and results were as followed.(1) Study on the effect of L-cysteine(Cys) interference on alloxan method and the development of applicable detection technology for the enzymatic synthesis of GSH. Interference experiment was designed to discuss the causes of complex interference phenomenon for the first time. H2O2 formed by the reaction between GSH, Cys and alloxan, and –SH from Cys contributed to the interference on the detection of GSH. With the design of interference model and compensation, a novel anti-interference detection technology, i.e. alloxan method with anti-interference compensation(AM-AC) was established coupled with automatic data processing. The results showed that the GSH concentration-absorbance standard equation of AM-AC was y=3.0629x+0.0001, R2=0.9999; when Cys concentration was 4 times that of GSH, the recoveries of GSH and Cys were 95%-100% and 78%-120% respectively by verification. Compared with the usual alloxan method, the new method shows a significant increase in anti-interference characteristics and accuracy for detection of the GSH content and semi-quantitative analysis of Cys.(2) To choose the optimal yeast addition amount and reaction time by tracking experiment. Maximum of GSH yield as detection index, yeast addition amount of 1.5 g and reaction time of 5 h were identified as the optimal conditions.(3) Research on the optimization experiment of precursor formula in the reaction liquid. With the test index of GSH yield and precursor conversion rate, design the experiments of single factor and orthogonal test. The results showed that for the influence of GSH yield, Cys was extremely significant, Glu, Gly were significant, and the weight order was Cys>Glu>Gly; and for the influence of precursor conversion rate, Cys was significant, Glu, Gly was not significant, and the weight order was Cys>Glu>Gly. It was verified and concluded that the optimal precursor formula was 4 mM Cys, 12 mM Glu and 50 mM Gly, and its conversion rate increased 112.17% than that of control(Glu 20 mM、Cys 15 mM and Gly 40 mM).(4) To promote GSH synthesis, H2O2 stress was brought in reaction liquid. In experiment of one-point H2O2 addition, 1-3 mM was a promotion concentration for the enzyme catalytic synthesis of GSH during 2-4 h reacted, but 0 h was the inappropriate time for the conversion rate. Design strategy experiment with one, two or three-point addition. The results showed that the optimum strategy was adding at 4 h to 1 mM H2O2 in reaction liquid, and its conversion rate had an average increase of 34.94% compared with that of the blank groups.