Purification And Characterisation Of Diamine Oxidase From Alfalfa

Amine oxidase widely exists in various organisms, and participates the biogenic amines metabolism. Monoamine oxidase is the combination enzyme which is Cu2+?Fe2+ and phosphatide. Polyamine oxidase is a monomeric soluble protein and catalyzed oxidation of polyamine. The discovered polyamine oxidase mainly exists in Legumes and Gramineae. Oxidizing the diamine to be catalytic is called diamine oxidase, and it is a kind of enzyme which can oxidize histamine, putrescine and cadaverine to be catalytic, and it also can regulate bioamines in circulating levels in the organization, diamine oxidase catalyzes the oxidation of amines to produce the corresponding aldehyde, hydrogen peroxide and other substances. In recent years, with the deepening of the research on diamine oxidase, in the fields of activity determination technique, immunohistochemistry and molecular biology the diamine oxidase's application have a certain progress.This paper using Alfalfa seedlings as tested materials, diamine oxidase activities were studied in different growth conditions, different growth stages and different parts of Alfalfa seedlings. Also to study the purification conditions, relative molecular mass, substrate specificity and thermal stability. First, under the condition of different illumination cultivation of Alfalfa seeds. Determination of the diamine oxidase activity which is influenced by light conditions. Second, gathering different growth period of Alfalfa seedlings and the different parts of diamine oxidase activity were determined. The crude enzyme was isolated and purified by sephadex G-100?DEAE-cellose and hydroxyapatite column.The purified enzyme was examined with polyacrylamide gel electrophoresis and calculated the relative molecular mass.The diamine oxidase activities were determined at different temperature 30??80? and determination the thremal stability.the putrescine?spermine?benzylamine?cadaverine were taken as substrates for the activity determination of diamine oxidase and study the substrate specificity. The results shows as follows:1.Alfalfa growth under different light conditions,the enzyme activity which cultured in dark(35.38 nKat) were higher than that in the light(8.89 nKat).2.Different growth period of Alfalfa seedlings (or not germinate seeds) enzyme activity were different.The diamine oxidase are not be measured in the unmalted seeds and soaking seeds after 12 hours. Incubating on the third day, the enzyme activity get the peak value (33.06nKat),and then began to decline. Incubating on the sixth day the activity dropped to 12.50 nKat.3.Hypocotyl, cotyledon and roots of Alfalfa seedlings were respectively detected diamine oxidase activity.And hypocotyl (78.91nKat)>root (24.82 nKat)>cotyledon (16.73 nKat).4.When ammonium sulfate precipitation the protein in crude extract, The best segmented salting-out of ammonium sulfate saturation is 20%-50%.5.The extraction and purification results showed that the enzyme activity of 458mL homogenate crude extract was 225.98 nKat, protein content is 77 mg. After the sulfate ammonium precipitation, the enzyme activity was 135.21 nKat, protein content in 13mg, recovery rate was 60%. From sephadex G-100 elution, enzyme activity was 104.65 nKat, protein content is 8 mg, recoveiy rate was 46%. From DEAE Sephacel column elution, enzyme activity was 85.20 nKat, protein content of 3mg, recovery rate was 37%. From Hydroxyapatite column elution, the enzyme activity was 59.27 nKat, protein content was 0.7 mg, recovery rate was 26%.6.The purity enzyme was detected by SDS-PAGE,In non-denaturing polyacrylamide gel electrophoresis appeared one protein bands, the relative molecular mass of about 110 kDa, and in denaturing polyacrylamide gel electrophoresis appeared two protein bands, the relative molecular mass of about 55 kDa and 40 kDa.7.The results of the study of thermal stability show that The results show that under 50? the catalytic activity of diamine oxidase of alfalfa was the highest and when the temperature of 80?, enzyme activity almost completely lost, lowering the temperature can not be restored the enzyme activity.8.Different substrates on diamine oxidase specificity studies showed that substrate for putrescine and enzyme activity the highest and reached 287.09 nKat, putrescine strongly to Alfalfa diamine oxidase specificity.Biogenic amine has very important effect to the life, and the amine oxidase also have important implications for the life. Not only to the organism has physiological significance, but also for various industries such as food, spices and medical has interest value. So it's very important to research on amine oxidase. Now the results can provide basic data for the future research.