Study On The Properties And Structure Of Glutamate Decarboxylase In Mung Bean

γ-aminobutyric acid,also known as 4-aminobutyric acid（GABA）,has many physiological functions and these physiological functions are very important.Glutamate decarboxylase（GAD,EC 4.1.1.15）is a catalytic enzyme of GABA and can catalyze L-glutamic acid to produceγ-aminobutyric acid.Preparation of GABA using plant tissues to metabolize GAD is a good method and approach.Through research we can find that mung bean has higher GAD vitality.The GAD in the paper is isolated and purified from mung beans.The main research is the GAD activity,GAD separation and purification,and the influence of temperature,p H,metal ions and reagents on GAD and the structure of GAD.To explore the activity of GAD in mung bean,and to provide a theoretical basis for further use of mung bean GAD enrichment to generate GABA.In the paper,GABA was derivatized by FDNB pre-column derivatization.This reaction used glutamate decarboxylase to catalyze the decarboxylation of L-glutamic acid to generateγ-aminobutyric acid.The activity of GAD in mung beans was detected.Derivatives were detected at 360 nm,and samples were separated using an HPLC Lichrospher C 18 column to obtain GABA content and GAD activity in mung bean tissue;ammonium sulfate precipitation method and DEAE-Cellulose ion exchange chromatography were established,Sephadex G-100 chromatography separation and purification of GAD in mung bean;the effect of different conditions on the precipitation effect of ammonium sulfate salting out GAD protein samples,and the enzymatic properties of purified mung bean GAD（Including temperature,p H,chemical reagent metal ions）;the molecular composition and molecular weight were detected by Native-PAGE electrophoresis,size exclusion high performance liquid chromatography（SE-HPLC）and mass spectrometry;the secondary structure of GAD was studied by Fourier transform infrared spectroscopy（FTIR）,using fluorescence spectroscopy to determine the tertiary structure,so as to study and understand the structure of mung bean GAD.The study mainly reached the following conclusions:The optimal precipitation conditions for the precipitation effect of ammonium sulfate salting out GAD protein samples are: the optimal sulfate concentration is 50%,the optimal precipitation time is 12 h,p H 6.0;the optimal conditions for DEAE-Cellulose ion exchangechromatography are: 30 The mung bean GAD（concentration 0.4 mg/m L）extract of p H 7.0 was applied to the column at a flow rate of 1.5 m L/min at30 ℃,the sample volume was 6 m L;Sephadex G-100 chromatography with dextran gel was the best The conditions are: sample volume 5 m L,elution with distilled water,elution flow rate 0.2 m L/min,absorbance at 280 nm;after separation and purification of GAD in mung bean,two isozymes are obtained with a molecular weight of 155 k Da（GAD1）and 75 k Da（GAD2）dimer,we can know the purification fold of GAD1 and GAD2,the specific activity of the enzyme and the enzyme recovery rate.The enzymatic properties of the two purified GADs can be obtained,and the best active p H values of GAD1 and GAD2 are p H 6.1 and p H 5.5,respectively,and the optimal temperature is40 ℃.GAD2 has a wider and stable p H range and temperature Scope;KI,Mg SO4,Ag NO3 and SDS have an inhibitory effect on mung bean GAD activity;Tween 80,Ca2+,Cu2+ and low concentration coenzyme PLP can promote mung bean GAD activity,but Fe²⁺ can only increase GAD2 activity.Through structural studies on GAD,it can be seen that GAD1 has a higher content of acidic amino acids,while GAD2 has a higher content of basic amino acids.Both mung bean GAD isozymes are composed of polar amino acids and non-polar amino acids in similar amounts.By studying the secondary structure of GAD through Fourier Transform Infrared Spectroscopy（FTIR）,it can be known that higher content of ordered structure was found in GAD2,and the higher content of α-helix and β-sheet determines the higher stability of GAD2.And fluorescence analysis shows that GAD1 has a more flexible and exposed molecular structure,while GAD2 has a more compact and conserved conformation.In summary,the optimal precipitation conditions were determined: the optimal sulfate concentration was 50%,the optimal precipitation time was 12 h,p H 6.0;the conditions for separation and purification were optimized and determined,and two GADs were separated,GAD1 and GAD2 were Dimer and higher enzyme activity;through enzymological studies,it can be seen that GAD2’s p H and temperature have wider stability,but only a small part of metal ions have an effect on GAD2 activity,which reduces its activity,and it is found in GAD2 higher The content of the ordered structure can also further explain its stability;through fluorescence analysis,changes in p H and temperature will cause structural changes in the enzyme and reduce enzyme activity,and GAD2’s more compact and conservative conformation further illustrates itsstability.In addition,the metal ion Ca²⁺ participates in the binding of the calmodulin binding domain of GAD,which induces a “buried” dense structure,and the expansion of GAD induced by SDS destroys the enzyme activity.A relatively high GAD activity was found in mung beans.The use of mung beans to prepare GABA has good prospects,and mung beans are cheap and easily available.The research content has far-reaching significance for the deep processing of mung beans and the production of new GABA.At the same time,it can also enrich the understanding of plant GAD and theoretically enrich mung beans.GABA provides support.