Preparation And Identification Of Black Soybean Lipoxygenase

Black soybeans are leguminous plants with black soybean seed,and are also a traditional medicinal and edible plant.The ancient books records that black soybeans could have some effects about black hair,kidney,detoxification,prolong life etc.Modern medicine also shows that black beans can be making the hair be black,detoxification,improving eyesight,soften blood vessels,such as the treatment of infertility.On the point of nutritional,black soybeans are rich in protein,fat,vitamins,trace elements and other nutrients.At the same time,it has a variety of biological active substances,such as isoflavone,saponin,black soybean pigment.We extract and purify black soybean lipoxygenase(bsLOX)containing?-glucosidase activity and investigate the properties of degradation of anthocyanins and enzymatic activity.Purification of bsLOX was carried out by buffer extraction,amm-onium sulfate precipitation,dialysis,cation exchange chromatographya-nd gel filtration chromatography.The activities of lipoxygenase and?-glucosidase on bsLOX were analyzed by using sodium linoleate and p-Nitrophenyl?-D-glucopyranoside(pNPG)as substrates,respectively.Different temperatures and pH effect on the lipoxygenase and?-gluco-sidase activity of bsLOX.Fluorescence spectrum were used to study the effect of on the structure of bsLOX.And three different anthocya-nidins effect on the lipoxygenase activity of bsLOX.HPLC wereused to study whether bsLOX could degrade anthocyanins(Cyanidin-3-O-Glucoside,Petunidin-3-O-glucoside,Delphinidin-glucoside),and three different anthocyanidins effect on the?-glucosidase activity of bsLOX.The results showed bsLOX was a kind of lipoxygenase proteins with?-glucosidase activity from black soybean seeds.Purified bsLOX indicated a single band via SDS-PAGE,and the molecular weight of bsLOX was 95 kDa.Analyzing of enzymes activity showed that purif-ied protein had a lipoxygenase activity about 13 U,and produced nit-ro phenol using PG as substrate when detecting at 405 nm.Fluoresce-nce spectrum showed anthocyanins(cyanidin,petunidin,elphinidin)could combine with bsLOX.HPLC results showed that bsLOX could degra-de anthocyanins(Cyanidin-3-O-glucoside,Petunidin-3-O-glucoside)into aglycone anthocyanidins(cyanidin,petunidin)and glucose,in turn,anthocyanidins could inhibit the activity of bsLOX.LOX in black so-ybean may be a kind of protein with a variety of enzymatic functions,while anthocyanidins may be a natural LOX inhibitor.