Extraction And Purification Of Lectin From Golden Crown Beans And Evaluation Of Its Coagulation Activit

Northeast snap bean is a characteristic small vegetable garden product in Heilongjiang Province.These beans not only reveal good taste,but also contain a variety of amino acids,dietary fiber,vitamins and minerals,which are very popular.In addition,the beans also contain some anti-nutritional substances,such asα-amylase inhibitors and lectins,which also have important medical value.Our research group has extractedα-amylase inhibitor from Golden Crowned snap beans previously,and this study was intended to extract lectin from it and study its properties.In this study,the seeds of Golden Crown snap beans were sampled at the mature stage,which was subjected to salting-out using 1.5%Na Cl to prepare the crude extract of protein,and then the protein was precipitated with 80%ammonium sulfate.The extraction and purification of the precipitated protein were carried out through molecular exclusion chromatography combined with ion exchange chromatography.In molecular exclusion chromatography,dextran gel G75 was used as filler,PBS was used as the eluent,at the flow rate of 0.5 m L/min.In the ion exchange chromatography,DEAE-A50 was used as filler,and the PB was used sas buffer solution.50 mmol/L and 100 mmol/L Na Cl in PB was used as eluent,and the flow rate was 0.3 m L/min.Next,the lectin properties were evaluated.2%rabbit red blood cells were selected to determine the total activity of lectin;The molecular weight of lectin was determined by SDS-PAGE and Native-PAGE.Three monosaccharides and three disaccharides were used to study the specific affinity of lectin.The stability of lectin against heat and p H was also studied.Finally,the functionality of lectin was studied,including the activity determination of lectin on 2%bovine blood,sheep blood,chicken blood and rabbit red blood cells and the antibacterial activity determination of lectin on Staphylococcus aureus,Escherichia coli and Listeria monocytogenes.Results:The content of protein after ammonium sulfate precipitation was90.33±5.56 mg/g.The sugar content was 2.58±0.42 mg/g,and the agglutination activity was 6.382×10~2 HU.The content of protein was 25.71±0.91 mg/g and the agglutination activity was 4.19×10~6HU after molecular exclusion chromatography(using dextran gel G75)and anion exchange chromatography(using DEAE-A50).The lectin from the seed of the Golden Crown bean was a tetramer composed of four subunits with a molecular weight of 44 k Da,and its molecular weight was about 170 k Da.In the monosaccharide binding experiment,the lectin had the strongest binding ability with D-fructose.The number of agglutinating pores was 6 at the lowest sugar concentration of 6.25 mmol/L;In the disaccharide binding experiments,lectin showed the best binding capacity with lactose,and the number of agglutination pores was 9.It also showed that the combination of D-fructose and lactose with lectin was better than that of D-fructose.In the stability test,the lectin maintained a high agglutination activity at 30-50℃.The agglutination activity was high over the range of p H 3.0～7.0.In the functional experiment,the lectin had almost no coagulation activity on bovine blood,and the number of agglutinating pores on sheep blood,chicken blood and rabbit blood were 8,10 and 9 respectively.The lectin has no obvious bacteriostatic effect on Listeria monocytogenes,Staphylococcus aureus and Escherichia coli.