| Laetiporus sulphureus is a kind of edible and medical fungi.When laetiporus sulphureus is young,it is edible and delicious.When it grows old,it is cheeselike and inedible,but it can be used as a functional medicine.Fungi lectins have defense and anti-tumor functions,and also have the effects of promoting mitosis and regulating cell and intercellular substance-mediated response.However,there are still few reports at home and abroad about laetiporus sulphureus lectin,especially its extraction,purification and structure.For the purpose of this article,the extraction and purification of lectins from laetiporus sulphureus are extablished,and its structure identification and physicochemical property are also investigated.First,the extraction parameters of laetiporus sulphureus lectin were studied.Sodium chloride concentration,liquid-solid ratio,extraction temperature,extraction time and extraction pH value were selected in single-factor experiments.Based on the single factor experiments,the four factors like sodium chloride concentration,liquid-solid ratio,extraction temperature,extraction time were selected to do four factors and three levels orthogonal experiment.When the sodium chloride concentrations of 0 mol/L,liquid-solid ratio of 30:1(mL/g),extraction temperature of 15?,extraction time of 10 h,extraction pH of 7.5 were adopted,the agglutination activity of laetiporus sulphureus lectin reached 0.5870 UH/mL and the extraction rate of protein was 0.576%.Then the laetiporus sulphureus lectin was isolated and purified.Target protein was precipitated with ammonium sulfate and the best ammonium sulfate saturation range of target protein was 20%?70%.The sample was further chromatographed on a DEAE-cellulose-52 column to obtain five peaks,D1?D2?D3?D4 and D5.The agglutinability of D3 was the highest with 28 and the yield of D3 reached 9.2%.D3 was chromatographed on a Sephadex G-100 column to obtain a peak of D3S1,whose agglutinability reached 28 and the recovery of 20.3%,respectively.It was a glycoprotein containing 4.81%sugar by pheonol-sulfuric acid reaction.According to the results of SDS-PAGE electrophoresis and Native-PAGE electrophoresis,the molecular weight of the lectin was 52.0 KDa,which was composed of the subunits of molecular weight of 35.1 KDa and 23.7 KDa,and the purity reached the level of electrophoresis.Using potassium bromide tablet method,the purified laetiporus sulphureus lectin was analized by fourier transform infrared spectroscopy of laetiporus sulphureus lectin.Laetiporus sulphureus lectin showed typical absorption peaks of protein in the range of 4000 cm-1?400 cm-1,further indicating it was a kind of protein.Amino acid composition analysis demonstrated that laetiporus sulphureus lectin had about 17 amino acids and there were plentiful of aspartic acid,glutamic acid,leucine and threonine in it,whose contents were 9.89%,9.41%,6.27%and 6.04%,respectively.The content of cysteine and methionine were 1.00%and 1.33%,respectively.The circular dichroism spectra revealed that the content of alpha helix,beta fold,beta-rotation was 24.2%,33.8%,19.3%and 34.3%.Using Atomic Force Microscope(AFM)to observe the surface morphology of laetiporus sulphureus lectin,it was an ellipsoid protein monomer with a diameter of 100?200 nm and a height of 5-20 nm.Sugar chains were not found.The physicochemical property of laetiporus sulphureus lectin was described.Lactose had inhibitory effect on its agglutination activity,and the minimum concentration of completely inhibiting the agglutinability was 100 mmol/L.However,glucose,D-fructose and D-mannose had no inhibitory effect on it.Laetiporus sulphureus lectin remained relatively stable in an acid environment,while unstable in an alkaline environment.When the temperature reached 80?,its agglutinability reached 0,which suggested that the laetiporus sulphureus lectin was unstable under higher heat.The strongest positive ion inhibiting its agglutinability was Fe3+,followed by Al3+and Ca2+.Mg2+,Zn2+and Mn2+had no effect on its agglutinability. |
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