Study On Enzymatic Characteristics Of Glutamate Decarboxylase And Succinic Semialdehyde Dehydrogenase In Germinated Tartary Buckwheat

Fagopyrum tartaricum(Fagopyrum Tartaricum(L.)Gaertn),known as tartary buckwheat,is rich in nutrients.In particular,it contains more phenolic substances such as rutin,quercetin,morin hydrate,kaempferol and so on.It has the functions of softening blood vessels,improving microcirculation,clearing away heat and detoxification,activating blood circulation and removing blood stasis,lowering blood sugar,lowering blood lipid and strengthening insulin.But it also has antinutritional factors such as protease inhibitors and so on.Antinutritional factors of Fagopyrum tartaricum can be eliminated or degraded by germination.It can also accumlation of y-aminobutyric acid(GABA)and other functional substances.Glutamate decarboxylase(GAD)is a ratelimiting enzyme for GABA synthesis,succinic semialdehyde dehydrogenase(SSADH)is the last step of GABA metabolic pathway.It catalyzes the irreversible conversion of succinic semialdehyde to succinic acid.Studying the enzymatic characteristics of GAD and SSADH is beneficial to GABA enrichment.In this paper,the optimum conditions for SSADH activity determination of germinated tartary buckwheat were optimized by response surface methodology.The enzymatic properties of GAD and SSADH in germinated tartary buckwheat were studied.The main achievement were as follows:1.Ractional precipitation of GAD extraction solution from germinated tartary buckwheat by(NH4)2SO4(40%～60%).After precipitation,the GAD purification multiple of germinated tartary buckwheat was 4.30 times,the specific activity of GAD was 2.02 U/mg,and the recovery yield of enzyme activity was 78.36%.The optimum reaction temperature for GAD of germinated tartary buckwheat is 40℃,it can maintaining higher thermal stability at 30～50℃.The optimum reaction pH for GAD of germinated tartary buckwheat is 5.7,which can maintain higher activity at pH 5.0～8.0.Cu2 +and Mg2+had no significant effect on GAD activity of germinated tartary buckwheat,but there was no significant difference between them(P>0.05).K+、Na+and SDS process significantly reduced GAD activity(P<0.05),but there was no significant difference among the three process(P>0.05).The Km and Vmax of GAD for L-Glu in germinated tartary buckwheat were 0.0197 mol/L and 3.34 g/min.2.Ractional precipitation of SSADH extraction solution from germinated tartary buckwheat by(NH4)2SO4(50%～80%).After precipitation,the SSADH purification multiple of germinated tartary buckwheat was 3.18 times,the specific activity of SSADH was 84.33 ×103U/mg,and the recovery yield of enzyme activity was 63.78%.On the basis of single factor experiment,the effects of temperature,pH and SSA concentration on SSADH activity of germinated tartary buckwheat were studied by response surface methodology.The results show that temperature and SSA had extremely significant effects on SSADH activity of germinated tartary buckwheat.pH had significant effects on SSADH activity.The interaction between temperature and pH is significant(P<0.05).The optimum conditions for SSADH activity determination of germinated tartary buckwheat were as follows:temperature 30.8℃,pH 8.7,SSA concentration 0.3 mmol/L.Under these conditions,the SSADH activity of germinated tartary buckwheat was 346±9.61 nmol/min.Analysis of variance and validation tests showed that the determinant coefficient of the model is 0.9892(P<0.001).It shows that the model is extremely significant.Correction factor R2adj=0.9752.It indicated that the model could explain 97.52%of the response value and accurately predict the SSADH activity of germinated tartary buckwheat.3.The optimum reaction temperature and pH of SSADH for Germinated tartary buckwheat were 30.8℃ and 8.7 respectively.The thermal stability of SSADH for Germinated tartary buckwheat was the best at 25℃,and the half-life of SSADH was 180.00 min,which was better than 64.44 min and 25.56 min for Germinated tary buckwheat at 30℃ and 35℃.At pH 7.6,8.1 and 8.6,the stability of SSADH in germinated tartary buckwheat was almost the same,with half-lives of 34.19 min,32.72 min and 35.83 min,respectively.At pH 9.1,the stability of SSADH in germinated tartary buckwheat was poor,with half-lives of 23.04 min.Cu2+,Co2+,Ni2+ have activation effect on SSADH activity of germinating tartary buckwheat.Among them,Cu2+ had the greatest influence(P<0.05),which was 1.21 times as much as that of the control group.Zn2+,Mn2+,Na+ can inhibited SSADH activity of germinating tartary buckwheat,and Zn2+has the greatest inhibitory effect on SSADH activity(P<0.05).Mg2+,Fe2+,Fe3+,Ca2+ had no significant effect on SSADH activity of germinated tartary buckwheat.There was no significant difference between them(P>0.05).The Km and Vmax of SSADH for SSA in germinated tartary buckwheat were 0.24 mmol/L and 583.24 nmol/min.The Km and Vmax of SSADH for NAD+in germinated tartary buckwheat were 0.64mmol/L and 454.55 nmol/min.