高等学校化学学报 ›› 2014, Vol. 35 ›› Issue (11): 2329.doi: 10.7503/cjcu20140492

• 有机化学 • 上一篇    下一篇

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

张洪涛1, 朱莉3, 张爽2, 詹晓北1()   

  1. 1. 江南大学生物工程学院糖化学与生物技术教育部重点实验室, 无锡 214122
    2. 江南大学生物工程学院食品科学与技术国家重点实验室, 无锡 214122
    3. 江苏瑞光生物科技有限公司, 无锡 214125
  • 收稿日期:2014-05-27 出版日期:2014-11-10 发布日期:2014-10-15
  • 作者简介:联系人简介: 詹晓北, 男, 博士, 教授, 主要从事多糖的发酵法生产研究. E-mail:xbzhan@yahoo.com
  • 基金资助:
    国家自然科学基金(批准号: 31201384, 3117164)、 国家博士后科学基金(批准号: 2012M20996)、 江苏省博士后基金和江苏省资助博士后基金(批准号: 1301011B)、 国家建设高水平大学公派研究生项目(批准号: 2008679005)、 高等学校学科创新引智计划(批准号: 111-2-06)和中央高校基本科研业务费专项资金(批准号: JUSRP1007)资助

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

ZHANG Hongtao1, ZHU Li3, ZHANG Shuang2, ZHAN Xiaobei1,*()   

  1. 1. Key Laboratory of Carbohydrate Chemistry and Biotechnology of Ministry of Education, School of Biotechnology,2. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China
    3. Jiangsu Rayguang Biotech. Co, Ltd., Wuxi 214125, China
  • Received:2014-05-27 Online:2014-11-10 Published:2014-10-15
  • Contact: ZHAN Xiaobei E-mail:xbzhan@yahoo.com
  • Supported by:
    † Supported by the National Natural Science Foundation of China(Nos.31201384, 3117164), the Postdoctoral Science Foundation of China(No.2012M20996), the Jiangsu Postdoctoral Science Foundation(No.1301011B), the China Scholarship Council Program(No.2008679005), the “111” Project(No.111-2-06) and the Fundamental Research Funds for the Central Universities of China(No.JUSRP1007)

摘要:

将香菇多糖和0.02 mol/L三氟乙酸(TFA)溶液按照1 mg:200 μL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.

关键词: 香菇多糖, 葡聚寡糖, 水解机制, 电喷雾碰撞诱导串联质谱

Abstract:

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 at 2, 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-gluco-oligosaccharides 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 gluco-oligosaccharides in large-scale and it also expand the gluco-oligosaccharides library for exploring protein-carbohydrate interaction using carbohydrate microarray.

Key words: Lentinan, Gluco-oligosaccharide, Mechanism of hydrolysis, Electrospray ionization-collision induce dissociation-tandem mass spectrometry(ESI-CID-MS/MS)

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