灰树花多糖D-组份的分离纯化及理化性质的研究
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摘要
本文通过对灰树花多糖的提取、纯化和结构的研究，确定了多糖D-组份(G-D)提取和纯化的工艺条件，分析了灰树花多糖D-组份(G-D)的分子结构，并建立了灰树花多糖D-组份(G-D)的检验方法。
1．研究确定了灰树花粗多糖的一些分析方法，测得灰树花粗多糖中多糖含量为28.9％，蛋白质含量为35.61％，β-葡聚糖含量在3.3％～3.4％之间，粗多糖溶液的pH值为6.3。
2．通过对市场上几个主流的相关产品的分析，确立了G-D的HPLC检测方法。
3．确定了G-D分离纯化的工艺：分级醇沉中乙醇的浓度分别为45％和80％；选用DEAE Sepharose Fast Flow作为离子交换剂，离子交换时的最佳pH为7.2；为了进一步提纯G-D，又选用了Sepheracyl S-400作为凝胶过滤介质，同时确定了洗脱液的pH值为7.2，离子强度为1.0mol／L；最终获得纯度很高的G-D，得率为0.45％。它是一种棕褐色、易吸潮、水溶性好且水溶液吸光度为—62.5°的固体粉末。经过分析发现，它的多糖含量为79.5％，蛋白质含量为16.2％。
4．选用木瓜蛋白酶作为制备G-D糖肽时的水解酶，获得所需糖肽；通过β-消除反应和肼解反应发现，G-D中糖肽间的连接方式中没有或者只有极少量的O-型糖肽键，但一定含有N-型糖肽键；将肼解后的糖链进行完全酸水解，并分析它的单糖组成情况，发现它含有D-葡萄糖、D-半乳糖、D-甘露糖，比例为1.8：0.4：29.4。通过傅立叶红外分析验证了G-D是一种糖蛋白，并发现它含有β-D-吡喃甘露糖。~1H NMR和~3C NMR分析发现，G-D中存在β-(1→6)连接方式。所以，G-D中主要是β-(1→6)-D-吡哺甘露糖苷键。
5．为验证本检测方法的实用性，对庆元灰树花粗多糖和迁西灰树花粗多糖中G-D含量进行比较发现，庆元灰树花中的G-D含量是迁西的1.78倍。
6．本文初步探讨了FPLC分离灰树花多糖中其它糖蛋白组份的方法，有一定的分离效果，但要分离到色谱纯的组分，分离柱类型和洗脱条件还需进一步选择优化。
本研究创新点在于：
1．建立了简便实用的灰树花多糖D-组份(G-D)的检测方法：
硕士论文
灰树花多糖D·组份的分离纯化及理化性质的研究
2
3
.确立了G一D简便、可靠的提取与纯化方法;
.分析了G一D中糖肤间的连接方式，确认它没有或只含有少量的O一型糖肤
键，但是含有N一型糖肤键;
4.经完全酸水解，发现G一D糖链的单糖组成，除了甘露糖外还存在少量的葡
萄糖和半乳糖;
5.采用FT-RI检测和NMR分析发现，G一D是一种含有p一(l一6)一D一毗喃
甘露糖普键的糖蛋白
Systematic studies on the extraction , purification and structural characterization of Grifola frondosa polysaccharides and its D-fraction were carried out in the presented dissertation. Some new methods and points were developed on the basis of thesis of these studies. The results and creative point are described as follows:
1. Found the content of polysaccharides in the crude polysaccharides is 28.9%,and the content of protein is 35.61% by Kjeldahl determination, and the content of 3 -glucan is 3.3%~3.4%.
2. Built a simple detecting method about G-D by HPLC According to some scientific reports and the analysis of the products on the market which are described mainly constituted with G-D.
3. The optimal concentration of ethanol are 45% and 80% in the two alcohol -insoluble stages; DEAE Sepharose Fast Flow is suitable for G-D ion exchange chromatography with the eluant at pH 7.2 and Sepheracyl S-400 is appropriate for the further purification of G-D with 1mol/L NaCl eluant at pH 7.2. G-D is a brownish black , deliquescent and diffluent powder ,whose specific rotatory power is -62. 5 . Found the content of polysaccharides in G-D is 79.5%,and the content of protein is 16.2%.
4. Using papain, get G-D glycopeptide. By β -elimination and hydrazinolysis,
found there is no or a few 0-type glycopeptide bond but N-type glycopeptide bond in G-D. After degraded by H2SO4 there are three kinds of monosaccharide: D-glucose, D-galactose, D-mannitose,and the ratio isl.8:0.4: 29.4. The FT-RI and 1H ,13C NMR spectrum indicated that there are β -(1→6)-D-mannopyranose in G-D chains.
5.By compared the content of G-D in the Grifola frondosa crude polysaccharides from Qingyuan and Qianxi, find that which in the former is 1.78 times as in the later.
6.Adopting FPLC to separate other glucoproteins in the crude polysaccharides can make every component reach the separation of the certain degree. But the type of FPLC columns and the elution should be further optimized.
the innovations that originally studies is embodied in the following several points:
1. A simple , convenient and practical detection method of G-D have been set up.
2. A simple and convenient, reliable extraction and purification method of G-D have been established
3. The way of joining between polysaccharide chains and peptide chains in G-D have been analysed, confirm that it may contain a few O-type glucoprotein, but must contain N-type glucoprotein.
4. The monoses of G-D were analysed ,it contains outside a large amount of mannose , A small amount of glucose, galactose exist yet.
5. the units of G-D chains was detected, it mostly contains β -(1→ 6)-D-mannopyranose .
1．研究确定了灰树花粗多糖的一些分析方法，测得灰树花粗多糖中多糖含量为28.9％，蛋白质含量为35.61％，β-葡聚糖含量在3.3％～3.4％之间，粗多糖溶液的pH值为6.3。
2．通过对市场上几个主流的相关产品的分析，确立了G-D的HPLC检测方法。
3．确定了G-D分离纯化的工艺：分级醇沉中乙醇的浓度分别为45％和80％；选用DEAE Sepharose Fast Flow作为离子交换剂，离子交换时的最佳pH为7.2；为了进一步提纯G-D，又选用了Sepheracyl S-400作为凝胶过滤介质，同时确定了洗脱液的pH值为7.2，离子强度为1.0mol／L；最终获得纯度很高的G-D，得率为0.45％。它是一种棕褐色、易吸潮、水溶性好且水溶液吸光度为—62.5°的固体粉末。经过分析发现，它的多糖含量为79.5％，蛋白质含量为16.2％。
4．选用木瓜蛋白酶作为制备G-D糖肽时的水解酶，获得所需糖肽；通过β-消除反应和肼解反应发现，G-D中糖肽间的连接方式中没有或者只有极少量的O-型糖肽键，但一定含有N-型糖肽键；将肼解后的糖链进行完全酸水解，并分析它的单糖组成情况，发现它含有D-葡萄糖、D-半乳糖、D-甘露糖，比例为1.8：0.4：29.4。通过傅立叶红外分析验证了G-D是一种糖蛋白，并发现它含有β-D-吡喃甘露糖。~1H NMR和~3C NMR分析发现，G-D中存在β-(1→6)连接方式。所以，G-D中主要是β-(1→6)-D-吡哺甘露糖苷键。
5．为验证本检测方法的实用性，对庆元灰树花粗多糖和迁西灰树花粗多糖中G-D含量进行比较发现，庆元灰树花中的G-D含量是迁西的1.78倍。
6．本文初步探讨了FPLC分离灰树花多糖中其它糖蛋白组份的方法，有一定的分离效果，但要分离到色谱纯的组分，分离柱类型和洗脱条件还需进一步选择优化。
本研究创新点在于：
1．建立了简便实用的灰树花多糖D-组份(G-D)的检测方法：
硕士论文
灰树花多糖D·组份的分离纯化及理化性质的研究
2
3
.确立了G一D简便、可靠的提取与纯化方法;
.分析了G一D中糖肤间的连接方式，确认它没有或只含有少量的O一型糖肤
键，但是含有N一型糖肤键;
4.经完全酸水解，发现G一D糖链的单糖组成，除了甘露糖外还存在少量的葡
萄糖和半乳糖;
5.采用FT-RI检测和NMR分析发现，G一D是一种含有p一(l一6)一D一毗喃
甘露糖普键的糖蛋白
Systematic studies on the extraction , purification and structural characterization of Grifola frondosa polysaccharides and its D-fraction were carried out in the presented dissertation. Some new methods and points were developed on the basis of thesis of these studies. The results and creative point are described as follows:
1. Found the content of polysaccharides in the crude polysaccharides is 28.9%,and the content of protein is 35.61% by Kjeldahl determination, and the content of 3 -glucan is 3.3%~3.4%.
2. Built a simple detecting method about G-D by HPLC According to some scientific reports and the analysis of the products on the market which are described mainly constituted with G-D.
3. The optimal concentration of ethanol are 45% and 80% in the two alcohol -insoluble stages; DEAE Sepharose Fast Flow is suitable for G-D ion exchange chromatography with the eluant at pH 7.2 and Sepheracyl S-400 is appropriate for the further purification of G-D with 1mol/L NaCl eluant at pH 7.2. G-D is a brownish black , deliquescent and diffluent powder ,whose specific rotatory power is -62. 5 . Found the content of polysaccharides in G-D is 79.5%,and the content of protein is 16.2%.
4. Using papain, get G-D glycopeptide. By β -elimination and hydrazinolysis,
found there is no or a few 0-type glycopeptide bond but N-type glycopeptide bond in G-D. After degraded by H2SO4 there are three kinds of monosaccharide: D-glucose, D-galactose, D-mannitose,and the ratio isl.8:0.4: 29.4. The FT-RI and 1H ,13C NMR spectrum indicated that there are β -(1→6)-D-mannopyranose in G-D chains.
5.By compared the content of G-D in the Grifola frondosa crude polysaccharides from Qingyuan and Qianxi, find that which in the former is 1.78 times as in the later.
6.Adopting FPLC to separate other glucoproteins in the crude polysaccharides can make every component reach the separation of the certain degree. But the type of FPLC columns and the elution should be further optimized.
the innovations that originally studies is embodied in the following several points:
1. A simple , convenient and practical detection method of G-D have been set up.
2. A simple and convenient, reliable extraction and purification method of G-D have been established
3. The way of joining between polysaccharide chains and peptide chains in G-D have been analysed, confirm that it may contain a few O-type glucoprotein, but must contain N-type glucoprotein.
4. The monoses of G-D were analysed ,it contains outside a large amount of mannose , A small amount of glucose, galactose exist yet.
5. the units of G-D chains was detected, it mostly contains β -(1→ 6)-D-mannopyranose .
引文
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