以酵素轉換結合高效能液相層析串聯質譜法建立甘露聚醣之特徵結構分析平台

Abstract

甘露聚醣依其醣結構骨幹可分為四大類群，包含直線型甘露聚糖、葡萄甘露聚糖及分支型半乳甘露聚糖、半乳葡萄甘露聚糖。由於甘露聚醣具有醣組成與大分子量的結構複雜性，為了鑑別其骨幹的類型，需建立一個β-(1,4)鍵結甘露聚醣的結構重建模型。本研究利用三類骨幹特徵的直線型甘露聚糖、半乳甘露聚糖及葡萄甘露聚糖，以Endo-β-(1,4)-mannanase酵素將其轉換為保留骨幹特徵的甘露寡醣，再以高效能石墨化碳液相層析串聯質譜法將寡糖分子的異構物分離，及高能碰撞誘導解離後的二次質譜斷片模式進行特徵鍵結組成的結構解析，推導出3種直線型(1→4)鍵結同質的甘露寡醣、4種直線型(1→4)鍵結異質的葡萄甘露寡醣、及5種具有(1→6)鍵結分支的異質甘露寡醣，並輔以酵素轉換率進行分支型甘露聚醣骨幹特徵的推論。本研究所發展之重建模型可用於分支型骨幹的甘露聚醣結構特徵鑑別。

Mannans are classified into four subfamilies, namely, linear mannan, glucomannan, galactomannan and galactoglucomannan. Because of complexity in sugar composition and high molecular weight, it is harder to identify backbone structure in mannans. Linear mannan, galactomannan and glucomannan were conducted for this study so as to identify backbone structure in mannans. Endo-β-(1,4)-mannanase was used to convert β-(1,4)-linkage backbone mannan into characteristic mannooligosaccharides, and then high performance porous graphitic carbon chromatography tandem mass spectrometry (PGC HPLC-MS/MS) was used to analyze oligosaccharide isomers and linkage fragmentation pattern. 3 kinds of linear homo-mannooligosaccharides, 4 kinds of linear hetero-mannooligosaccharides, and 5 kinds of branching hetero-mannooligosaccharides were determined in this study. The results showed that backbone of galactomannan can be identified by branching percentage. This approach can be used as backbone structure analysis in galactomannan.