Isolation Of Lectin-like Protein From The Microcystis In Dianchi Lake And The Research On Properties

Microbial lectins are proteins (or glycoproteins) from microorganism, other than antibodies and enzymes, which bind specifically and reversibly to carbohydrates, resulting in cell agglutination or precipitation of glycoconjugates. The lectins are of important value for medicine, biology, agriculture and other fields. Its function is involved in identifying in cell recognition, toxicity determining factor, anti-virus and so on.In this study, we isolated and cultured Microcystis from cyanobacteria in Dianchi Lake. Then lectin and phycocyanin were purified from Microcystis. Some characteristics of lectin and phycocyanin were studied comparatively.The lectin from the Microcystis was isolated and purified with sequential steps, i.e. cell disruption, precipitation by (NH₄) ₂SO₄, DEAE Sephadex A-25 ion exchange chromatography and Sephadex G-75 chromatography. The purified lectin showed high ability to agglutinate rabbit erythrocyte, the minimum agglutinating concentrations (MAC) is 8．59μg/mL, and MAC both are 17．19μg/mL for some Thermus cell and human B erythrocyte. The apparent molecular weight of the lectin was determined by sodium dodecyl suflate-polyacrylamide gel electrophoresis to be 24．0KDa. The carbohydrate content of 3．91% suggested that the lectin was glycoprotein. The Carbohydrate inhibition experiments of agglutinating activity indicated the activity was inhibited by glucose, galactose, D-xylan, D-sorbitol. The agglutinating activity was relatively insensitive to temperatures at 20℃～50℃, but dropped rapidly at 60℃or the higher temperature. The lectin appeared the stable agglutinating activity with a wide pH range, especially in pH 5．0～9．16．The effects of the other factors were found to be diverse on the agglutinating activity. For example, experiments showed the agglutinating activity depended on two divalent cations: Ca²⁺ and Mg²⁺. However, denaturant such as urea and SDS for the activity only affected to a certain extent. The experiment revealed that some organic solvent greatly effect on the agglutinating activity, such as methyl alcohol and n-hexane. The best of the agglutinating activity of lectin was preserved in 4℃by specific illumination test. The lectin had good anti-oxidizing activity, and the specific activity is 3．98U/mg. Then inhibition of the lectin to HIV_-IIIB in vitro was weak by using MTT method. In addition, the lectin maybe contains tyrosine, tryptophan and phehylalanine by fluorescent spectrum, and some residue of tryptophan may in the hydrophobic envioment. The specific conjugated saccharide of the lectin might be galactose by analysis.With the use of cell disruption, precipitation by (NH₄) ₂SO₄, DEAE Sephadex A-25 ion exchange chromatograph and Sephadex G-75 chromatography, the phycocyanin was similarly isolated and purified from Microcystis. The phycocyanin has relative molecular weight range of less than 14．4KDa by SDS-PAGE electrophoresis, and contains 1．07% neutral saccharide. The agglutinating experiments showed that phycocyanin agglutinated rabbit erythrocyte (MAC=28．2μg/mL) and human O erythrocyte specifically. The phycocyanin was observed that its agglutinating activity was inhibited by glucose and sucrose. The activity was more stable at 20℃～30℃, and it was completely lost at 70℃or above temperature. The chemical denaturation experiments indicated that the agglutinating activity highest data was in pH 4．0, and the activity was least unchangeable at pH 5．0 and maximally unchangeable at pH 8．0．The anti-oxidizing activity of phycocyanin per unit was 6．44U/mg.Through the comparison of the essential characteristics of lectin and phycocyanin, results showed that the lectin had better stability in temperature and pH treatment, of slightly higher molecular weight or carbohydrate content, better the agglutinating activity for rabbit erythrocytes than the phycocyanin. However, the anti-oxidizing activity and illumination stability of lectin was not as good as phycocyanin.