基于ESI-CID-MS/MS离子碎片峰特征的香菇多糖水解机制及系列香菇寡糖的制备

Mechanism of Lentinan Hydrolysis Based on ESI-CID-MS/MS and Preparation of Full Series of Oligosaccharides

将香菇多糖和0.02 mol/L三氟乙酸(TFA)溶液按照1 mg∶200μL比例混合并于100℃水解,然后利用快速蛋白液相色谱(FPLC)技术对分别水解2,4,6 h的水解产物中不同聚合度寡糖的组成与丰度进行分析,从而确定最佳水解时间;用确定的最佳时间对水解残留多糖重复水解3次,直至多糖沉淀消失为止,然后分别对水解产物进行FPLC分离并依次收集每批次的七糖.对七糖进行ESI-CID-MS/MS分析,结合β-1,3-二糖、七糖及β-1,6-葡聚寡糖二糖的ESI-CID-MS/MS质谱特征,可获得每次水解产物中寡糖的结构信息,进而获得香菇多糖的水解机制;最后用P4凝胶柱对水解产物进行色谱分离,获得具有不同聚合度的全序列寡糖.结果表明,香菇多糖的最佳水解时间为4 h,在不同批次的水解过程中香菇寡糖的结构由β-1,3/1,6-寡糖过渡为纯β-1,3-寡糖.

To acquire the mechanism of lentinan hydrolysis and full series of gluco-oligosaccharides from hydrolyzed lentinan,the hydrolysis time of lentinan treated with 0. 02 mol /L trifluoroacetic acid（ TFA） at 100 ℃was optimized through analysis the distribution of different degree of polymerization（ DP） oligosaccharides at2,4,and 6 h. And then lentinan hydrolysis was repeated another three times until no precipitation can be observed under optimal hydrolysis time. The mass spectrometry character of 7-mer oligosaccharides from all the four batch hydrolysis products was analyzed using ESI-CID-MS /MS based on the mass spectrometry character of 7-mer β-1,3-and β-1,6-gluco-oligosaccharides,and acquiring a hydrolysis mechanism. Finally,all the four batch hydrolysis products were combined and separated with P4 column and full series of gluco-oligosacchaides from 2-mer to 13-mer were acquired. The optimal time for lentinan hydrolysis was 4 h,and β-1,3 /1,6-glucooligosaccharides in hydrolysis lentinan was observed at the first batch,and then the amount of β-1,3 /1,6-gluco-oligosaccharides reduced with the increasing of hydrolysis times,and there only observed β-1,3-gluco-oligosaccharides at the fourth hydrolysis products. This results also suggested that the triple helix structure zone containing β-1,3 /1,6-linkage of lentinan is loose,but the only containing β-1,3-linkage triple helix structure zone in lentinan is tight. The theory acquired in this work will supply useful information for preparation glucooligosaccharides in large-scale and it also expand the gluco-oligosaccharides library for exploring protein-carbohydrate interaction using carbohydrate microarray.