银耳多糖提取纯化、结构特征及溶液性质研究
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摘要
以银耳子实体为原料,采用热水煮提、醇沉法提取银耳粗多糖,经酶-Sevage法去蛋白、DEAE-52纤维素离子交换层析和Sephacryl S-400凝胶过滤层析纯化后得到银耳均一多糖TFP1,用苯酚-硫酸法、间羟联苯-硫酸法检测其总糖、糖醛酸等组分的含量；PMP衍生化后反相高效液相色谱测单糖组成,结合红外光谱和核磁共振图谱分析银耳多糖一级结构特征；利用尺寸排阻色谱-激光光散射(SEC-LLS)、碘=碘化钾实验、刚果红实验、粘度检测和原子力显微镜技术对多糖溶液性质进行初步探讨。
组分分析表明精制银耳多糖TFP1总糖含量为81.9%,糖醛酸含量为6.9%,不含蛋白质和核酸；单糖组成分析、红外光谱及核磁共振图谱结果表明TFP1主要由甘露糖、葡萄糖醛酸、木糖和岩藻糖四种单糖组成(含少量葡萄糖),其物质的量比约为5.78：0.7：1.1：1,TFP1是分子构型为α=型且含有O-乙酰基结构的酸性杂多糖。
SEC-LLS检测计算出TFP1在NaCl溶液中的重均分子量(Mw)为5.832×106Da,多分散系数(Mw/Mn)为1.005,均方根旋转半径(z1/2)为191.3 nm,说明TFP1是分子量较大且糖链为刚性链的均一多糖。由Huggins方程和Kraemer方程计算的TFP1的特性粘度[η]=1508.3 mL/g,所以TFP1在25℃的0.1 mol/LNaCl溶液中具有较高的刚性,分子链较为伸展。碘-碘化钾实验中300-700 nm紫外-可见光吸收图谱中565 nm处没有最大吸收值,说明TFP1是具有多分支或支链的大分子。随着碱浓度升高,刚果红与银耳多糖形成的络合物最大吸收波长相应减小,但相对于刚果红本身最大吸收波长减少明显缓慢,说明其没有三螺旋结构。由于自身带电荷、分子内作用力和多支链等原因在原子力显微镜下观察到TFP1的形貌呈球状。
综上,银耳多糖TFP1是以甘露糖为主链,主要由四种单糖残基(含少量葡萄糖)组成,含有O-乙酰基结构且具有多个分支的酸性均一杂多糖,分子构型为a-型。溶液性质研究表明TFP1是分子量较大且伸展的刚性链,不存在三螺旋结构。
A water-soluble polysaccharide was extracted from Tremella fuciformis Berk. Crude Tremella fuciformis polysaccharide was gained by boiling in hot-water, precipitating using ethanol, removing proteins using papain-Sevage method. Then the crude polysaccharide was applied to DEAE-52 cellulose colume and further purified through Sephacryl S-400 column, followed by dialysis, lyopilization. The resulting sample was named as TFP1. Total sugar and uronic acid contents of TFP1 were determined by phenol-sulfuric acid method and m-hydroxydiphenyl combined with sulfuric acid method using glucose and glucuronic acid as standard respectively. UV spectrum showed that no protein or nucleic acid presented in TFP1. The results of the PMP precolumn derivatization HPLC analysis, FT-IR spectroscopy and NMR spectrum showed that TFP1 was an acidic heteropolysaccharide containing mannose, glucuronic acid, xylose and fucose with the molar ratio of 5.78:0.7:1.1:1(containing a small amount of glucose), and the spectrums indicated that anomeric carbon of TFP 1 was a-configuration. The Mw, polydispersity index and root-mean square radius of polymer calculated from Zimm plot was 5.832×106Da,1.005,193.1 nm in NaCl solution respectively, which indicated that TFP1 was a homogeneous and rigid chain macromolecule. The intrinsic viscosity([η]) was used to determined by both of the Huggins and Kraemer functions was 1508.3 mL/g which indicated that the polymer molecules was considered to occupy a relatively large hydrodynamic volume in NaCl solution. I2-KI assay also suggested that many branched chains attached to the backbone chain which was composed of mannose residues. After Congo red dye was added to sample at various concentration of alkali, the variation curves of maximum absorption wavelength of mixture decreased gradually with increasing of NaOH concentration and no triple helical structure was detected in TFP1. Atomic force microscopy was used to characterize surface topology and visualize the orientation and spatical distribution of molecules absorbed to mica surface. TFP1 was observed (?)mpact chains conformation unexpectedly, and it was probable that the structure characteristics of GlcUA residue, abundant hydrogen bonds and hyperbranch tend to make macromolecule form spherical conformation.
In brief, TFP1 was an acidic and homogeneous heteropolysaccharide mainly composed of four monosaccharide residues and the anomeric carbons were a-configuration. Solution properties research proved that the hyperbranched polysaccharide existed in the solution as an extended rigid chain but without triple helical conformation.
组分分析表明精制银耳多糖TFP1总糖含量为81.9%,糖醛酸含量为6.9%,不含蛋白质和核酸；单糖组成分析、红外光谱及核磁共振图谱结果表明TFP1主要由甘露糖、葡萄糖醛酸、木糖和岩藻糖四种单糖组成(含少量葡萄糖),其物质的量比约为5.78：0.7：1.1：1,TFP1是分子构型为α=型且含有O-乙酰基结构的酸性杂多糖。
SEC-LLS检测计算出TFP1在NaCl溶液中的重均分子量(Mw)为5.832×106Da,多分散系数(Mw/Mn)为1.005,均方根旋转半径(
综上,银耳多糖TFP1是以甘露糖为主链,主要由四种单糖残基(含少量葡萄糖)组成,含有O-乙酰基结构且具有多个分支的酸性均一杂多糖,分子构型为a-型。溶液性质研究表明TFP1是分子量较大且伸展的刚性链,不存在三螺旋结构。
A water-soluble polysaccharide was extracted from Tremella fuciformis Berk. Crude Tremella fuciformis polysaccharide was gained by boiling in hot-water, precipitating using ethanol, removing proteins using papain-Sevage method. Then the crude polysaccharide was applied to DEAE-52 cellulose colume and further purified through Sephacryl S-400 column, followed by dialysis, lyopilization. The resulting sample was named as TFP1. Total sugar and uronic acid contents of TFP1 were determined by phenol-sulfuric acid method and m-hydroxydiphenyl combined with sulfuric acid method using glucose and glucuronic acid as standard respectively. UV spectrum showed that no protein or nucleic acid presented in TFP1. The results of the PMP precolumn derivatization HPLC analysis, FT-IR spectroscopy and NMR spectrum showed that TFP1 was an acidic heteropolysaccharide containing mannose, glucuronic acid, xylose and fucose with the molar ratio of 5.78:0.7:1.1:1(containing a small amount of glucose), and the spectrums indicated that anomeric carbon of TFP 1 was a-configuration. The Mw, polydispersity index and root-mean square radius of polymer calculated from Zimm plot was 5.832×106Da,1.005,193.1 nm in NaCl solution respectively, which indicated that TFP1 was a homogeneous and rigid chain macromolecule. The intrinsic viscosity([η]) was used to determined by both of the Huggins and Kraemer functions was 1508.3 mL/g which indicated that the polymer molecules was considered to occupy a relatively large hydrodynamic volume in NaCl solution. I2-KI assay also suggested that many branched chains attached to the backbone chain which was composed of mannose residues. After Congo red dye was added to sample at various concentration of alkali, the variation curves of maximum absorption wavelength of mixture decreased gradually with increasing of NaOH concentration and no triple helical structure was detected in TFP1. Atomic force microscopy was used to characterize surface topology and visualize the orientation and spatical distribution of molecules absorbed to mica surface. TFP1 was observed (?)mpact chains conformation unexpectedly, and it was probable that the structure characteristics of GlcUA residue, abundant hydrogen bonds and hyperbranch tend to make macromolecule form spherical conformation.
In brief, TFP1 was an acidic and homogeneous heteropolysaccharide mainly composed of four monosaccharide residues and the anomeric carbons were a-configuration. Solution properties research proved that the hyperbranched polysaccharide existed in the solution as an extended rigid chain but without triple helical conformation.
引文
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