Purification And Characterisation Of Monoamine Oxidase From Wheat

Monoamine oxidase（MAO）is an enzyme that can oxidize and decompose primary amine and some secondary amine.The research has found that this enzyme mainly exists on the outer membrane of mammalian mitochondria.There are two types of MAO isoenzymes in mammals,namely MAO-A and MAO-B;The coding genes for these two isoenzymes are located in the X chromosome and are independent of each other,which enable them to demestracte different substrate specificity.At present,there is relatively little research on the separation,purification,and enzymatic properties of wheat MAO both domestically and internationally.This project isolated and purified MAO from wheat,and indicates the factors that affect its extraction.The experimental results are as follows:（1）The effect of light conditions on MAO activity in wheat seedlings was studied,and it was found that MAO activity in wheat seedlings grown under dark conditions was higher than that under light conditions;（2）A comparative analysis was conducted on the activity of MAO in different parts of wheat seeds and seedlings.It was found that the order of MAO activity in wheat seeds,young shoots,and roots was:seedling roots>seeds>young shoots;（3）Optimization of the extraction,separation and purification process of MAO from wheat seedlings was studied,and it was found that the optimal p H of the extraction solution was 7,and the optimal liquid-solid ratio was 3:1.However,the effect of extraction time on MAO activity was not significant;（4）Study on the separation and purification process of MAO crude enzyme solution from wheat seedlings.After centrifugation,ammonium sulfate precipitation,dextran gel G-75chromatography,dialysis,DEAE cellulose ion exchange chromatography and other operations,MAO with high purity was obtained,the recovery rate was22%,and the specific enzyme activity was 100U/mg;（5）The reaction conditions and stability of MAO in wheat seedlings were studied,and it was found that the optimal reaction temperature was 37℃and the optimal reaction p H was 7.0.MAO has poor thermal stability and is prone to deactivation at high temperatures;（6）Studying the enzymatic properties of MAO in wheat seedlings,it was found that its Km value to benzylamine was8.40mmol/L,and it had almost no reaction to putrescine and spermine.Metal ions such as Fe²⁺,Fe³⁺,Cu²⁺have a strong promoting effect on the activity of MAO.Ca²⁺,Mg²⁺can slightly promote the activity of MAO,while K⁺,Na⁺,and Zn²⁺have no effect on the activity of MAO.Testing the inhibitory effects of inhibitors such as oxalic acid,citric acid,ascorbic acid,diglycidyl peptide,cinnamic acid,trans cinnamic acid,kojic acid,ferulic acid,4-ethylresorcinol,and sodium bisulfite on wheat MAO activity,it was found that oxalic acid,ascorbic acid,diglycidyl peptide,cinnamic acid,kojic acid,4-ethylresorcinol,and sodium bisulfite have a significant inhibitory effect on MAO activity,with ascorbic acid having the strongest inhibitory effect on MAO,The inhibitory effect can reach 90%.